(r20) Barrick Lab

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

*%RED%THIS PAGE IS CURRENTLY UNDER CONSTRUCTION - APOLOGIES FOR ANYTHING THAT'S STILL A BIT MESSY!%ENDCOLOR%* 

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
---+++ *4a. Choose your own qPCR Adventure*

     <img src="%ATTACHURLPATH%/qPCRflowchartcolor.png" alt="qPCRflowchartcolor.png" width='725' height='535' border="2" align="right" />

*Choices before starting*

In order to perform the qPCR that will answer your biological questions, you will first need to go through a few rounds of qPCR optimization. The amount of preliminary qPCR plates you will need to set up before you do your experimental qPCR will vary based on what you've done before. Refer to the flow chart on the right to figure out the path that you should take and then follow the corresponding link below to learn about the details of each step!
<br>

*What step are you on?*
   * [[PrimerEfficiencyqPCR][Primer Efficiency qPCR]] -- Determine the efficiency of the primers you designed
   * [[cDNAConcentrationqPCR][cDNA Concentration qPCR]] -- Determine the optimal concentration of cDNA to use in your qPCRs
   * [[RefGeneqPCR][Reference Gene qPCR]]  -- Determine the most consistent ref gene for your conditions
   * [[ExperimentalqPCR][Experimental qPCR]] -- Run your experiment

---+++ *4b. qPCR Reaction Setup*

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.

*Before you begin:*

*1.* 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. <br>
*2.* Calculate stock solutions of sybrgreen/primer and cDNA. Each well will be loaded with the following:

     <img src="%ATTACHURLPATH%/qPCR_mastermix.png" alt="qPCR_mastermix.png" width='207' height='161.6' align="left" />

*NOTES*
   * Both primers are included in the total volume here (i.e. 0.25ul each). 
   * I would recommend a 200nM final concentration for the primers (0.5ul of 2000nM working stock), but you can try out others if desired.
   * The degree of dilution for the cDNA will be determined in one of your initial qPCR runs.

<br style="clear:both" />

*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>
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
xlsx	qpcr_plate_setup.xlsx	r1	manage	13.1 K	2019-03-21 - 15:49	KateElston	qPCR plate template
Barrick Lab > ProtocolList > QPCR
Contributors to this topic
KateElston, SimonDAlton, JuliePerreau, DanielDeatherage
Topic revision: r20 - 2019-04-05 - 22:44:32 - Main.KateElston