Quantitative real time PCR for differential gene expression

The following is a comprehensive strategy for qPCR that works and is practically, mathematically and statistically accurate. It conforms to the MIQE guidelines, which should be reported in all qPCR experiments (Bustin et al, 2009). Recommendations on primer Tm, statistical outputs and graphical representations etc may differ from previous work or practices.

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. 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 ‘two primers, intercalating dye’.
  • Click on ‘customize your assay’. Up the optimal Tm to 63 and the minimum Tm to 60.
  • Increase the optimal amplicon length to 120bp.
  • Click ‘get assays’ and find a primer pair that closely matches the above conditions and for which both the primer Tms are equal. Click ‘view assay details’. 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.

2. RNA preparation

Prepare RNA as detailed elsewhere on the Wiki, ensuring that you include on column DNase digestion. NOTE: column digestion of DNA is not always 100% complete. If you find 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.

3. Reverse Transcription

Use the high capacity reverse transcription kit from ABI.

Per sample:

RNA

500ng – 1ug

H2O

Up to 14.2ul

10x buffer

2ul

dNTPS

0.8ul

Random primers

2ul

RTase

1ul

Conditions:

  • 25C 10’
  • 37C 120’
  • 85C 5’

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

4. qPCR

Choices before starting

There are two approaches to qPCR, the most efficient and cost effective will depend on what you are trying to do. If you are designing an assay for a transcript that you think you will use over and over again, use Method 2. If you are looking to answer one very specific question with few biological replicates (e.g. 5 per condition, 2 conditions), use Method 1.

The other choice to be made is between 384 and 96 well plates. If you’re using method 1, for every amplicon you are measuring, 18 wells need to be set aside for a standard curve. It makes a lot of sense to get used to loading a 5ul rxn in a 384 well plate. It is very cost and time effective to do so and it takes only a few plates worth of practice to get precise readings across technical replicates.

For 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.

General Guidelines on loading a plate

  • Plan your plate. In an excel spreadsheet, 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:

1x

sybrgreen

2.5ul

primers

0.5ul

cDNA

2ul

Both primers are included in the total volume here (i.e. 0.25ul each). The concentration is determined in the following sections.

The cDNA is diluted to a degree determined in following sections.

  • Mark up your qPCR plate with a Sharpie. See example below. This will help you to avoid ‘getting lost’ 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.

IMAG2516.jpg

  • Make the stock of sybrgreen and primers, keep in dark on ice until use.
  • Make cDNA stocks, keep on ice until use.
  • For loading it is useful to raise the plate so the wells can be seen more clearly:

IMAG2517.jpg

  • 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.
  • Load 2ul of cDNA of interest to each well, changing out tips between wells.
  • 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.
  • Peel away the serated edges of the film.
  • Spin down @ 1000rpm , 1 min.
  • Run on qPCR machine as per instructions during training.

4a. Method 1

PCR#1

Goals

  • Test that primers work
  • Verify single products via a melt curve analysis
  • Determine approximate Ct (cycle threshold) of PCR amplification
  • Test RNA for gDNA contamination
  • Typical plate setup for single reference gene and single target (you must test all candidate reference genes):

1

2

3

4

5

6

A

5

5

5

5

5

5

B

25

25

25

25

25

25

C

125

125

125

125

125

125

D

625

625

625

625

625

625

E

3125

3125

3125

3125

3125

3125

F

15625

15625

15625

15625

15625

15625

G

RNA

RNA

RNA

RNA

RNA

RNA

H

50nM

50nM

200nM

200nM

500nM

500nM

Ref

Target

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 experimental cDNA samples.
  • The RNA sample used is a pool of your RNA samples, diluted to the same extent as the 1:25 cDNA dilution (i.e. take 500ng of your pooled RNA, make up to 20ul (because your initial reverse transcription was 20ul) and then make a 1:25 dilution).

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).
  • PCR products that produce a single peak in your melt curve analysis
  • A dilution of cDNA that produces Ct values of between 13-30 cycles (If your Cts 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 >7 cycles later than the signal in your experimental sample.

PCR#2

Goals

  • Determine which of your reference genes you are going to normalize to

Typical plate setup for three candidate reference genes:

1

2

3

4

5

6

7

8

9

A

C1BR1

C1BR1

C1BR1

C1BR1

C1BR1

C1BR1

C1BR1

C1BR1

C1BR1

B

C1BR2

C1BR2

C1BR2

C1BR2

C1BR2

C1BR2

C1BR2

C1BR2

C1BR2

C

C1BR3

C1BR3

C1BR3

C1BR3

C1BR3

C1BR3

C1BR3

C1BR3

C1BR3

D

C1BR4

C1BR4

C1BR4

C1BR4

C1BR4

C1BR4

C1BR4

C1BR4

C1BR4

E

C1BR5

C1BR5

C1BR5

C1BR5

C1BR5

C1BR5

C1BR5

C1BR5

C1BR5

F

C2BR1

C2BR1

C2BR1

C2BR1

C2BR1

C2BR1

C2BR1

C2BR1

C2BR1

G

C2BR2

C2BR2

C2BR2

C2BR2

C2BR2

C2BR2

C2BR2

C2BR2

C2BR2

H

C2BR3

C2BR3

C2BR3

C2BR3

C2BR3

C2BR3

C2BR3

C2BR3

C2BR3

I

C2BR4

C2BR4

C2BR4

C2BR4

C2BR4

C2BR4

C2BR4

C2BR4

C2BR4

J

C2BR5

C2BR5

C2BR5

C2BR5

C2BR5

C2BR5

C2BR5

C2BR5

C2BR5

ref gene 1

ref gene 2

ref gene 3

C1 = CONDITION 1

C2 = CONDITION 2

BR# = BIOLOGICAL REPLICATE #

Conditions

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

What you are looking for

  • At least 2 and preferably 3 amplicons that show no significant difference between control and experimental conditions. The standard deviation of the Cts for all biological replicates should be low.
  • If you are seeing differences between replicates or perhaps conditions, you may be asking the question “how do I know if there’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!”. 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 (at most, 0.5 Ct) difference between biological replicates of the same condition, assuming that condition itself is reproducible. If the biological variation is truly large between replicates, you’ll have to pick the best you can.

PCR#3

Goals

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

1

2

3

4

5

6

7

8

9

A

5

5

5

5

5

5

5

5

5

B

25

25

25

25

25

25

25

25

25

C

125

125

125

125

125

125

125

125

125

D

625

625

625

625

625

625

625

625

625

E

3125

3125

3125

3125

3125

3125

3125

3125

3125

F

6250

6250

6250

6250

6250

6250

6250

6250

6250

G

C1BR1

C1BR1

C1BR1

C1BR1

C1BR1

C1BR1

C1BR1

C1BR1

C1BR1

H

C1BR2

C1BR2

C1BR2

C1BR2

C1BR2

C1BR2

C1BR2

C1BR2

C1BR2

I

C1BR3

C1BR3

C1BR3

C1BR3

C1BR3

C1BR3

C1BR3

C1BR3

C1BR3

J

C1BR4

C1BR4

C1BR4

C1BR4

C1BR4

C1BR4

C1BR4

C1BR4

C1BR4

K

C1BR5

C1BR5

C1BR5

C1BR5

C1BR5

C1BR5

C1BR5

C1BR5

C1BR5

L

C2BR1

C2BR1

C2BR1

C2BR1

C2BR1

C2BR1

C2BR1

C2BR1

C2BR1

M

C2BR2

C2BR2

C2BR2

C2BR2

C2BR2

C2BR2

C2BR2

C2BR2

C2BR2

N

C2BR3

C2BR3

C2BR3

C2BR3

C2BR3

C2BR3

C2BR3

C2BR3

C2BR3

O

C2BR4

C2BR4

C2BR4

C2BR4

C2BR4

C2BR4

C2BR4

C2BR4

C2BR4

P

C2BR5

C2BR5

C2BR5

C2BR5

C2BR5

C2BR5

C2BR5

C2BR5

C2BR5

Conditions

  • Numbers in rows A-F represent the dilution of a cDNA sample that is a pool of all experimental 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 triplicate higher than 0.2, and there is obviously an outlier, remove it. If the SD is higher than 0.2 and all three measurements are spread, keep all three.

Calculating relative quantities

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

example_calc_3.jpg

(the above example does not use amounts of cDNA i.e. ng, but sometimes it is easier to think about the following calculations by using ng. Just use the input into your reverse transcription and the subsequent dilutions to give you these numbers).

  • Using this standard curve, calculate the ng of each reference gene and target gene in each biological replicate.
  • Calculate the mean and standard deviation for target and reference genes for each condition.
  • For each condition:
    • Normalized target quantity = target gene mean / reference gene mean
    • If using multiple reference genes, divide by the geometric mean of the reference genes
    • ( standard deviation – follow the details on pg 40 through top of page 42 in ABI bulletin attached to this page)
  • Fold change = normalized target quantity (condition 1) / normalized target quantity (condition 2)
  • Plot this fold change as log2(fold change).

4b. Method 2

Topic attachments
I Attachment Action Size Date Who Comment
pdfpdf 1125331_ABI_-_Guide_Relative_Quantification_using_realtime_PCR.pdf manage 495.6 K 14 Feb 2017 - 21:16 Main.SimonDAlton  
jpgjpg IMAG2516.jpg manage 666.1 K 09 Feb 2017 - 21:29 Main.SimonDAlton  
jpgjpg IMAG2517.jpg manage 810.3 K 09 Feb 2017 - 21:36 Main.SimonDAlton  
jpgjpg example_calc.jpg manage 79.3 K 09 Feb 2017 - 22:18 Main.SimonDAlton  
jpgjpg example_calc_2.jpg manage 109.3 K 09 Feb 2017 - 22:22 Main.SimonDAlton  
jpgjpg example_calc_3.jpg manage 155.3 K 09 Feb 2017 - 22:23 Main.SimonDAlton  
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Topic revision: r5 - 26 May 2017 - 19:16:24 - Main.SimonDAlton
 
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