(r17) QPCR < Lab

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---+ Quantitative real time PCR for differential gene expression

Quantitative real time PCR is a tool routinely used for the quantitation of target nucleic acids. This protocol outlines the steps you will need to take if you want to quantify the relative upregulation or downregulation of a specific gene product following RNAi, or some other change in the environmental condition of your sample. There are a variety of different ways to perform the analysis for quantitative real time PCR (qPCR), and these different analysis methods inform what sorts of optimizations to perform each round of qPCR. This protocol will walk you through the preparation of cDNA from your biological samples and then you will have the chance to choose your analysis method and subsequent qPCR reactions to set up.

*Read through this complete protocol the first time you perform a qPCR experiment*.  

This is not an easy procedure, and you will likely have to repeat your qPCR rounds a few times until you can achieve low standard deviations between technical replicates. As with most things though, the more you practice the better you'll get! (I would also recommend reading [[%ATTACHURL%/1125331_ABI_-_Guide_Relative_Quantification_using_realtime_PCR.pdf][this]] in-depth document to better understand the entire protocol.)

---++ *General Materials*
   * Primers (see below for design)
   * ABI high capacity reverse transcription kit with random primers (from LifeTech freezer in MBB supply room)
   * RNase/DNase free water
   * Sybrgreen 2x PCR master mix (from LifeTech freezer in MBB supply room)
   * ABI 384/96 well optical plates (ThermoFisher cat#4309849/cat#N8010560)
   * Thermaseal Transparent sealing films for Real-time PCR, (Excel Scientific cat#TS-RT2-100)
   * Training on the ABI qPCR machines in MBB (contact the Core for information).



---++ *Protocol*
   1. [[ #PrimerDesign][Primer Design]]
   1. [[ #RnaPrep][RNA Preparation]]
   1. [[ #RevTran][Reverse Transcription]]
   1. [[ #QuantPcr][qPCR]]

#PrimerDesign
---+++ 1. Primer Design

Design primers to your target(s) and four reference genes. You will eventually use _at least_ 2 of these reference genes.

Reference genes:

   * You will need to find multiple reference genes to normalize expression to.
   * These need to be stable across all the conditions (control and experimental) and this needs to be empirically determined by your own testing (see later).
   * Candidate genes might come from: 
      * Previous publications
      * Previous experience
      * Microarray compendiums; variance in expression can be determined for individual genes from thousands of samples across multiple conditions (e.g. Faith et al, 2008 Nucleic Acids Research, 10.1093/nar/gkm815).

Design process:

   * Go to http://www.idtdna.com/primerquest/home/index, enter your sequence and select &lsquo;two primers, intercalating dye&rsquo;.
   * Click on &lsquo;customize your assay&rsquo;. Up the optimal Tm to 63 and the minimum Tm to 60.
   * Increase the optimal amplicon length to 120bp.
   * Click &lsquo;get assays&rsquo; and find a primer pair that closely matches the above conditions and for which both the primer Tms are equal. Click &lsquo;view assay details&rsquo;. Hovering over the primer sequence will give you options to check secondary structure and BLAST for off target transcripts. Do this.
   * Order your favorite primer pair.

#RnaPrep 
---+++ 2. RNA Preparation

Prepare RNA as detailed [[http://barricklab.org/twiki/bin/view/Lab/RNAPrep][here]] or [[http://barricklab.org/twiki/bin/view/Lab/RNAPlantPrep][here]], ensuring that you include on column DNase digestion. NOTE: column digestion of DNA is not always 100% complete. If you find significant DNA contamination in your RNA samples (see later), you need to complete DNase treatment using DNase 1 and use a column to clean up the RNA from the reaction mix.

#RevTran
---+++ 3. Reverse Transcription

Use the high capacity reverse transcription kit from ABI.

Per sample:

| RNA | 100 ng &ndash; 500 ng (x 	&micro;l) |
| 10x buffer| 2 &micro;l |
| dNTPS | 0.8 &micro;l |
| Random primers | 2 &micro;l |
| RTase | 1 &micro;l |
| H2O | 14.2 &micro;l - x &micro;l |

Conditions:

   * 25°C &ndash; 10 min
   * 37°C &ndash; 120 min
   * 85°C &ndash; 5 min

Store cDNA at -20°C, or for longer durations, -80°C

#QuantPcr
---+++ 4. qPCR

*Choices before starting*

There are a few approaches to qPCR and we'll walk through the approaches based on your desired experimental design. The *only* thing that changes in each approach are the plate setups for each round of qPCR. This will change based on the optimization that you will need to perform for your specific experiment.

The general instructions below will guide you in setting up the *384 well reaction plate*. For some experiments it may make more sense to set up a 96 well plate (the machines themselves can handle both sizes), but If you're loading quite a few samples, it is <strong>very </strong>cost and time effective to do so and it takes only a few plates worth of practice to get precise readings across technical replicates.

If using a 384 well plate, load a 5ul rxn. For a 96 well, load 20ul. The following assumes a 5ul load in a 384 well plate. Scale appropriately.

*General Guidelines on loading a plate*

   * Plan your plate. In an excel spreadsheet ([[%ATTACHURL%/qpcr_plate_setup.xlsx][qPCR plate template here!]]), plan the layout of your plate. *Print it off*. This is important with large plates. See following sections for examples.
   * Calculate stock solutions of sybrgreen/primer and cDNA. Each well will be loaded with the following:

<table width="102" cellspacing="0" style="width: 101.5pt; margin-left: 4.65pt; border-collapse: collapse" cellpadding="0" border="0"> <tbody><tr style="height: 15pt"> <td width="61" valign="bottom" style="width: 60.5pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="41" valign="bottom" style="width: 41pt; padding: 0in 5.4pt; height: 15pt">

*1x*
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*sybrgreen*
</td> <td width="41" valign="bottom" style="width: 41pt; border: 1pt solid windowtext; padding: 0in 5.4pt; height: 15pt">

2.5ul
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*primers (200nM)*
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0.5ul
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*cDNA*
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2ul
</td> </tr> </tbody></table>

Both primers are included in the total volume here (i.e. 0.25ul each). I would recommend a 200nM final concentration for these (0.5ul of 2000nM working stock), but you can try out others if desired; as with most steps of qPCR everything can be optimized to suit your needs.

The degree of dilution for the cDNA will be determined in one of your initial qPCR runs.

*Basic Steps for Loading a qPCR plate*

*1.* Mark up your qPCR plate with a Sharpie. See example below. This will help you to avoid &lsquo;getting lost&rsquo; on your qPCR plate. Other tips: keep track of what wells you have visited by referring to your print out and by matching the tip you are taking from your tip box with the well on the plate.

<img width="500" alt="IMAG2516.jpg" src="/twiki/pub/Lab/QPCR/IMAG2516.jpg" />

*2.* Make the stock of sybrgreen and primers, keep in dark on ice until use. <br>
*3.* Make cDNA stocks, keep on ice until use. <br>
*4.* Adjust your plate and get ready to load! It is useful to raise the plate so the wells can be seen more clearly: <br>

<img width="500" alt="IMAG2517.jpg" src="/twiki/pub/Lab/QPCR/IMAG2517.jpg" />

*5.* Load 3ul of sybrgreen/primer mix to each well. You do not need to change tips between wells for the same mix, only between mixes. It is important to accurately load each well: 
   * Lower the tip to the bottom of the well
   * Lightly touch the bottom
   * Eject the total contents
   * Keep the plunger depressed as you pull out of the well.
   * Do not drag the tip up the sides of the well when pulling out.
   * *Use non-barrier tips!* For some reason, less fluid is retained in them between wells.
*6.* Load 2ul of cDNA of interest to each well, changing out tips between wells. <br>
*7.* Place an optical cover on the plate and firmly seal by running a hard, flat, clean edge over the top and around the edges of the outermost wells. <br>
*8.* Peel away the serated edges of the film. <br>
*9.* Spin down @ 1000rpm , 1 min. (there is a plate centrifuge in the qPCR machine room) <br>
*10.* Run on qPCR machine as per instructions during training. <br>

---++++ *4a. Method 1*

*PCR#1*

_Goals_

   * Test that primers work
   * Verify single products via a melt curve analysis
   * Determine what dilution of your cDNA you will use as template in subsequent reactions 
   * Test RNA for gDNA contamination

Typical plate setup for single reference gene and single target (you must test all candidate reference genes):

<table width="316" cellspacing="0" style="margin-left: 4.65pt; border-collapse: collapse" cellpadding="0" border="0"> <tbody><tr style="height: 15pt"> <td width="29" valign="bottom" style="width: 28.65pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="44" valign="bottom" style="width: 44.1pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *1* </p> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *2* </p> </td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *3* </p> </td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *4* </p> </td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *5* </p> </td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *6* </p> </td> </tr> <tr style="height: 15pt"> <td width="29" valign="bottom" style="width: 28.65pt; padding: 0in 5.4pt; height: 15pt">

*A*
</td> <td width="44" valign="bottom" style="width: 44.1pt; border: 1pt solid windowtext; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> <td width="45" valign="bottom" style="width: 45pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> </tr> <tr style="height: 15pt"> <td width="29" valign="bottom" style="width: 28.65pt; padding: 0in 5.4pt; height: 15pt">

*B*
</td> <td width="44" valign="bottom" style="width: 44.1pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> </tr> <tr style="height: 15pt"> <td width="29" valign="bottom" style="width: 28.65pt; padding: 0in 5.4pt; height: 15pt">

*C*
</td> <td width="44" valign="bottom" style="width: 44.1pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> </tr> <tr style="height: 15pt"> <td width="29" valign="bottom" style="width: 28.65pt; padding: 0in 5.4pt; height: 15pt">

*D*
</td> <td width="44" valign="bottom" style="width: 44.1pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> </tr> <tr style="height: 15pt"> <td width="29" valign="bottom" style="width: 28.65pt; padding: 0in 5.4pt; height: 15pt">

*E*
</td> <td width="44" valign="bottom" style="width: 44.1pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> </tr> <tr style="height: 15pt"> <td width="29" valign="bottom" style="width: 28.65pt; padding: 0in 5.4pt; height: 15pt">

*F*
</td> <td width="44" valign="bottom" style="width: 44.1pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">15625</p> </td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">15625</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">15625</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">15625</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">15625</p> </td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">15625</p> </td> </tr> <tr style="height: 15pt"> <td width="29" valign="bottom" style="width: 28.65pt; padding: 0in 5.4pt; height: 15pt">

*G*
</td> <td width="44" valign="bottom" style="width: 44.1pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt">

RNA
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RNA
</td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt">

RNA
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RNA
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RNA
</td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt">

RNA
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*H*
</td> <td width="44" valign="bottom" style="width: 44.1pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; padding: 0in 5.4pt; height: 15pt">

50nM
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50nM
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200nM
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200nM
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500nM
</td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt">

500nM
</td> </tr> <tr style="height: 15pt"> <td width="29" valign="bottom" style="width: 28.65pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="44" valign="bottom" style="width: 44.1pt; background: none 0% 0% repeat scroll #e6b8b7; padding: 0in 5.4pt; height: 15pt">

Ref
</td> <td width="45" valign="bottom" style="width: 45pt; background: none 0% 0% repeat scroll #b8cce4; padding: 0in 5.4pt; height: 15pt">

Target
</td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> </td> </tr> </tbody></table>

Numbers are cDNA dilution, Concentrations are [primer]

_Conditions_

   * Primer concentration stated above is the final concentration in the rxn.
   * The cDNA used for this is a pool of all your cDNA samples i.e. take 1ul of each RT reaction product, pool them, and make dilutions from there. _Why?_ You don't know the difference in expression between your experimental and control sample, or the variation in your biological replicates. If you just ran this plate using cDNA template from one reaction, you might end up choosing conditions suitable for that sample, but not suitable for the others. This limits that error.
   * The RNA sample used to test for gDNA contamination is a pool of all your RNA samples (just like you did for the cDNA pool in the step above). This pool is then diluted to the same extent as the 1:25 cDNA dilution i.e. take Xng of your pooled RNA (x = ng you inputted into RT), make up to 20ul (because your initial reverse transcription was 20ul) and then make a 1:25 dilution.

_What you are looking for_

   * PCR products that produce a single peak in your melt curve analysis
   * A dilution of cDNA that produces Cq values of between 13-30 cycles (If your Cqs are less than or greater than this, the assay can still work, but depending on machine, standard deviations can get a bit noisy if amplification happens too early or late.)
   * No contamination in RNA, or contamination that is &gt;5 cycles later than the signal in your experimental sample. For primers that are 100% efficient, each cycle represents a doubling of product. Therefore a difference of 7 cycles would be a difference in expression of 2^7 or 128 fold. 

*PCR#2*

_Goals_

   * Determine which of your reference genes you are going to normalize to.

_Why am I doing this?_

Reference genes need to be stable across your control and experimental samples in order to be useful. If expression between the two differs, you will be normalizing to two different values and that is worthless. This is a common omission. Reference genes must be validated.

Typical plate setup for three candidate reference genes:

<table width="438" cellspacing="0" style="margin-left: 4.65pt; border-collapse: collapse" cellpadding="0" border="0"> <tbody><tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="47" valign="bottom" style="width: 46.75pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *1* </p> </td> <td width="51" valign="bottom" style="width: 51.15pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *2* </p> </td> <td width="42" valign="bottom" style="width: 42.45pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *3* </p> </td> <td width="46" valign="bottom" style="width: 45.9pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *4* </p> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *5* </p> </td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *6* </p> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *7* </p> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *8* </p> </td> <td width="50" valign="bottom" style="width: 0.7in; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *9* </p> </td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt">

*A*
</td> <td width="47" valign="bottom" style="width: 46.75pt; border: 1pt solid windowtext; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR1
</td> <td width="51" valign="bottom" style="width: 51.15pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR1
</td> <td width="42" valign="bottom" style="width: 42.45pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR1
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C1BR1
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR1
</td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR1
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR1
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR1
</td> <td width="50" valign="bottom" style="width: 0.7in; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR1
</td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt">

*B*
</td> <td width="47" valign="bottom" style="width: 46.75pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR2
</td> <td width="51" valign="bottom" style="width: 51.15pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR2
</td> <td width="42" valign="bottom" style="width: 42.45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR2
</td> <td width="46" valign="bottom" style="width: 45.9pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR2
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR2
</td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR2
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR2
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR2
</td> <td width="50" valign="bottom" style="width: 0.7in; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR2
</td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt">

*C*
</td> <td width="47" valign="bottom" style="width: 46.75pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR3
</td> <td width="51" valign="bottom" style="width: 51.15pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR3
</td> <td width="42" valign="bottom" style="width: 42.45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR3
</td> <td width="46" valign="bottom" style="width: 45.9pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR3
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR3
</td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR3
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR3
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR3
</td> <td width="50" valign="bottom" style="width: 0.7in; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR3
</td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt">

*D*
</td> <td width="47" valign="bottom" style="width: 46.75pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR4
</td> <td width="51" valign="bottom" style="width: 51.15pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR4
</td> <td width="42" valign="bottom" style="width: 42.45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR4
</td> <td width="46" valign="bottom" style="width: 45.9pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR4
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR4
</td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR4
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR4
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR4
</td> <td width="50" valign="bottom" style="width: 0.7in; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR4
</td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt">

*E*
</td> <td width="47" valign="bottom" style="width: 46.75pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR5
</td> <td width="51" valign="bottom" style="width: 51.15pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR5
</td> <td width="42" valign="bottom" style="width: 42.45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C1BR5
</td> <td width="46" valign="bottom" style="width: 45.9pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR5
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR5
</td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C1BR5
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR5
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR5
</td> <td width="50" valign="bottom" style="width: 0.7in; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C1BR5
</td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt">

*F*
</td> <td width="47" valign="bottom" style="width: 46.75pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C2BR1
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C2BR1
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C2BR1
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C2BR1
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C2BR1
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C2BR1
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C2BR1
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C2BR1
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C2BR1
</td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt">

*G*
</td> <td width="47" valign="bottom" style="width: 46.75pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C2BR2
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C2BR2
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C2BR2
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C2BR2
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C2BR2
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C2BR2
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C2BR2
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C2BR2
</td> <td width="50" valign="bottom" style="width: 0.7in; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C2BR2
</td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt">

*H*
</td> <td width="47" valign="bottom" style="width: 46.75pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C2BR3
</td> <td width="51" valign="bottom" style="width: 51.15pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C2BR3
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C2BR3
</td> <td width="46" valign="bottom" style="width: 45.9pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C2BR3
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C2BR3
</td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C2BR3
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C2BR3
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C2BR3
</td> <td width="50" valign="bottom" style="width: 0.7in; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C2BR3
</td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt">

*I*
</td> <td width="47" valign="bottom" style="width: 46.75pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C2BR4
</td> <td width="51" valign="bottom" style="width: 51.15pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C2BR4
</td> <td width="42" valign="bottom" style="width: 42.45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C2BR4
</td> <td width="46" valign="bottom" style="width: 45.9pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C2BR4
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C2BR4
</td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C2BR4
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C2BR4
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C2BR4
</td> <td width="50" valign="bottom" style="width: 0.7in; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C2BR4
</td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt">

*J*
</td> <td width="47" valign="bottom" style="width: 46.75pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C2BR5
</td> <td width="51" valign="bottom" style="width: 51.15pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C2BR5
</td> <td width="42" valign="bottom" style="width: 42.45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

C2BR5
</td> <td width="46" valign="bottom" style="width: 45.9pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C2BR5
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C2BR5
</td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

C2BR5
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C2BR5
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C2BR5
</td> <td width="50" valign="bottom" style="width: 0.7in; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

C2BR5
</td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="47" valign="bottom" style="width: 46.75pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="51" valign="bottom" style="width: 51.15pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="42" valign="bottom" style="width: 42.45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="46" valign="bottom" style="width: 45.9pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="50" valign="bottom" style="width: 0.7in; padding: 0in 5.4pt; height: 15pt"> </td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="47" valign="bottom" style="width: 46.75pt; border: 1pt solid windowtext; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

ref gene 1
</td> <td width="51" valign="bottom" style="width: 51.15pt; border-width: 1pt medium; border-style: solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

</td> <td width="42" valign="bottom" style="width: 42.45pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt">

</td> <td width="46" valign="bottom" style="width: 45.9pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

ref gene 2
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: 1pt medium; border-style: solid none; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

</td> <td width="50" valign="bottom" style="width: 49.5pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #b7dee8; padding: 0in 5.4pt; height: 15pt">

</td> <td width="45" valign="bottom" style="width: 45pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

ref gene 3
</td> <td width="45" valign="bottom" style="width: 45pt; border-width: 1pt medium; border-style: solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

</td> <td width="50" valign="bottom" style="width: 0.7in; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt">

</td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="47" valign="bottom" style="width: 46.75pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="51" valign="bottom" style="width: 51.15pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="42" valign="bottom" style="width: 42.45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="46" valign="bottom" style="width: 45.9pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="50" valign="bottom" style="width: 0.7in; padding: 0in 5.4pt; height: 15pt"> </td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="140" colspan="3" valign="bottom" style="width: 140.35pt; padding: 0in 5.4pt; height: 15pt">

C1 = CONDITION 1
</td> <td width="46" valign="bottom" style="width: 45.9pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="50" valign="bottom" style="width: 0.7in; padding: 0in 5.4pt; height: 15pt"> </td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="140" colspan="3" valign="bottom" style="width: 140.35pt; padding: 0in 5.4pt; height: 15pt">

C2 = CONDITION 2
</td> <td width="46" valign="bottom" style="width: 45.9pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="50" valign="bottom" style="width: 0.7in; padding: 0in 5.4pt; height: 15pt"> </td> </tr> <tr style="height: 15pt"> <td width="17" valign="bottom" style="width: 17pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="231" colspan="5" valign="bottom" style="width: 231.25pt; padding: 0in 5.4pt; height: 15pt">

BR# = BIOLOGICAL REPLICATE #
</td> <td width="50" valign="bottom" style="width: 49.5pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="45" valign="bottom" style="width: 45pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="50" valign="bottom" style="width: 0.7in; padding: 0in 5.4pt; height: 15pt"> </td> </tr> </tbody></table>

_Conditions_

   * Use primer concentration and cDNA dilution determined in PCR#1

_What you are looking for_

   * Technical replicates with a standard deviation below 0.2 (this is arbitrary and most of your replicates will be below 0.1. If you do enough qPCR, you will eventually become obsessed with how low you can get this number).
   * At least 2 and preferably 3 amplicons that show no significant difference between control and experimental conditions. The standard deviation of the Cqs for all biological replicates should be low.
   * If you are seeing differences between replicates or perhaps conditions, you may be asking the question &ldquo;how do I know if there&rsquo;s really a difference, it could be something else like loading or RT-PCR efficiency? I am not controlling for any of these by normalizing to anything!&rdquo;. The answer is if you prepared good quality RNA and loaded exactly the same amount of RNA into a well-prepared reverse transcription, there should be very little (less than 1 Cq) difference between biological replicates of the same condition, assuming that condition itself is reproducible. If the biological variation is truly large between replicates, you&rsquo;ll have to pick the best you can.

*PCR#3*

_Goals_

   * Test hypothesis
   * Typical plate setup with 2 reference genes and a target gene:

<table width="355" cellspacing="0" style="width: 355pt; margin-left: 4.65pt; border-collapse: collapse" cellpadding="0" border="0"> <tbody><tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt"> </td> <td width="40" valign="bottom" style="width: 40pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *1* </p> </td> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *2* </p> </td> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *3* </p> </td> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *4* </p> </td> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *5* </p> </td> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *6* </p> </td> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *7* </p> </td> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *8* </p> </td> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right"> *9* </p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*A*
</td> <td width="40" valign="bottom" style="width: 40pt; border: 1pt solid windowtext; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: 1pt 1pt 1pt medium; border-style: solid solid solid none; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">5</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*B*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">25</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*C*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">125</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*D*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">625</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*E*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">3125</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*F*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">6250</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">6250</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">6250</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">6250</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">6250</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">6250</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">6250</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">6250</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="right" style="text-align: right">6250</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*G*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR1</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*H*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR2</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*I*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR3</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*J*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR4</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*K*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C1BR5</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*L*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR1</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR1</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*M*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR2</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR2</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*N*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR3</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR3</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*O*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR4</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR4</p> </td> </tr> <tr style="height: 15pt"> <td width="35" valign="bottom" style="width: 35pt; padding: 0in 5.4pt; height: 15pt">

*P*
</td> <td width="40" valign="bottom" style="width: 40pt; border-width: medium 1pt 1pt; border-style: none solid solid; border-image-source: none; border-image-slice: initial; border-image-width: initial; border-image-outset: initial; border-image-repeat: initial; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #e4dfec; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #dce6f1; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR5</p> </td> <td width="35" valign="bottom" style="width: 35pt; border-width: medium 1pt 1pt medium; border-style: none solid solid none; background: none 0% 0% repeat scroll #fcd5b4; padding: 0in 5.4pt; height: 15pt"> <p align="center" style="text-align: center">C2BR5</p> </td> </tr> </tbody></table>

_Conditions_

   * Numbers in rows A-F represent the dilution of a cDNA sample that is a pool of all samples. These values will be used to create standard curves to determine relative quantities of target between conditions.
   * Should also include a water control for each primer pair.
   * Primer concentrations and the dilution used for the experimental samples will be those determined from PCR#1.

_What you are looking for_

   * Technical replicates with a standard deviation below 0.2.
   * Outliers; if you have an SD for a technical triplicate higher than 0.2, and there is obviously an outlier, remove it. An example is if you have values of 23.34, 23.35 and 30.22, and the 30.22 amplification trace is clearly poor. If the SD is higher than 0.2 and all three measurements are spread, you must keep all three.

*Calculating relative quantities*

   * Plot log10(cDNA input) v Ct standard curves for each primer pair, e.g.:

<img width="750" alt="example_calc_3.jpg" src="/twiki/pub/Lab/QPCR/example_calc_3.jpg" />

   * Using this standard curve, calculate the concentration of each reference gene and target gene in each biological replicate.
   * Calculate the mean for target and reference genes for each condition.
   * For each condition: 
      * Normalized target quantity = target gene mean / average of reference gene means
   * Fold change = normalized target quantity (experimental) / normalized target quantity (control)
   * Plot this fold change as log2(fold change).
Attachments
Topic attachments
I	Attachment	History	Action	Size	Date	Who	Comment
pdf	1125331_ABI_-_Guide_Relative_Quantification_using_realtime_PCR.pdf	r1	manage	495.6 K	2017-02-14 - 21:16	SimonDAlton
jpg	IMAG2516.jpg	r1	manage	666.1 K	2017-02-09 - 21:29	SimonDAlton
jpg	IMAG2517.jpg	r1	manage	810.3 K	2017-02-09 - 21:36	SimonDAlton
jpg	example_calc.jpg	r1	manage	79.3 K	2017-02-09 - 22:18	SimonDAlton
jpg	example_calc_3.jpg	r1	manage	155.3 K	2017-02-09 - 22:23	SimonDAlton
xlsx	qpcr_plate_setup.xlsx	r1	manage	13.1 K	2019-03-21 - 15:49	KateElston	qPCR plate template
Topic revision: r17 - 2019-03-21 - KateElston