1

Q-PCR: Basic Protocol

by Jean-Denis Benazet, May 2009

I. General

Try to standardize the whole procedure for a complete experiment: the amount of tissue

processed, the amount of RNA retro-transcribed, the cDNA volume in each reaction, the

threshold value used for determining the Ct (if you do it manually). In general, nine

independent samples per tissue-type and genotype should be analyzed to get statistically

significant results. However, this may vary according to your exact experimental conditions.

II. Sample collection (mouse limb buds from E9.5 to E11.5)

If collecting other tissues, then please adapt this protocol accordingly:

1. Collect embryos in ice-cold PBS.

2. Dissect embryos individually in a separate dish. Change the PBS for each embryo.

Use clean and sharp forceps for dissection. Clean them regularly. Both forelimb buds

are collected as one sample.

3. Transfer the sample into a 1.5 ml Eppendorf tube. For limb buds, glass pipets are used

as the samples stick less to glass than to plastic.

4. Remove the maximum PBS using a Gilson P10 pipet. Cover the samples with

RNAlater (50-100µl). The samples have the tendency to float. Briefly centrifuge and

store at 4°C. The next day check all tubes and if samples still float recentrifuge tubes.

Store samples at -20°C for prolonged times. For more details consult the RNAlater

Users Manual.
 2

III. RNA isolation and purification

1. Remove the RNAlater using a Gilson (use the P10 for the last drops). Only use filter

tips.

2. Follow the instructions of the Micro RNeasy Kit from Qiagen for extracting RNA

from pairs of forelimb buds up to E11.5.

3. Add the RNA carrier (polyA-RNA provided with the kit) when working with E9.0-

E9.5 limb bud pairs. For more details consult the Users Manual.

4. To disrupt the cells, pass the solution containing the sample 15x through a 25G needle

using a 1ml syringe. Change syringe and needle for each sample.

5. Load the samples on the columns as described in the Users Manual.

6. Treat the samples with DNase on the column. Use an aliquot of DNase that is not too

old. For further details and elimination of the DNAse, see the Users Manual.

7. Store the purified total RNA at -80°C.

IV. cDNA synthesis

DO NOT SCALE UP!

The following 20-µl reaction volume can be used for 10 pg-4µg of total RNA or 10 pg-500ng

of mRNA.

1. Add the following components to a nuclease-free microcentrifuge tube:

1 µl of oligo(dT)20 (50µM); or 200-500 ng of oligo(dT)12-18

10 pg-5 µg total RNA or 10 pg-500 ng mRNA

1 µl 10 mM dNTP Mix (10 mM each dATP, dGTP, dCTP and dTTP at neutral pH)
 3

Sterile distilled water to a total volume of 13 µl

2. Heat mixture to 65ºC for 5 minutes and incubate on ice for at least 1 minute.

3. Collect the contents of the tube by brief centrifugation and add:

4µl 5x First Strand Buffer

1µl 0.1M DTT

1µl RNaseOUTTM Recombinant RNase Inhibitor (Cat. No. 10777-019),

40 units/µl). Note: When using less than 50 ng of starting RNA, the addition of

RNaseOutTM is essential.

1 µl of SuperscriptTM III RT (200 units/µl)*

*If generating cDNA longer than 5 kb at temperatures above 50ºC, the amount of

SuperscriptTM III RT may be raised to 400 U (2µl) to increase yield.

4. Mix by pipetting gently up and down.

5. Incubate at 50ºC for 60 minutes.

6. Inactivate the reaction by heating at 70ºC for 15 minutes.

7. The cDNA can now be used as a template for amplification in PCR. However,

amplification of some PCR targets (those > 1 kb) may require the removal of RNA

complementary to the cDNA. To remove RNA complementary to the cDNA, add 1 µl (2

units) of E. coli RNase H and incubate at 37ºC for 20 minutes.

8. Store cDNA at – 20ºC.

9. For further details see Users Manual.

V. Q-PCR reaction using Sybr Green

General: Vortex all solutions extensively before pipetting

 4

1. SybR green mix per reaction: 2µl of primer mix + 12.5 µl SybR green.

Primer Mix: 7.5µl of Forward and Reverse Primer stock (100µM) and

185ul EB (Qiagen); Endconc.: 3.75µM of each Primer

2. cDNA mix per reaction: 0.05 µl of cDNA in 10.5 µl of H 2O MilliQ (concentration for

one pair of limb buds at 10.5-10.75).

3. Distribute 14.5 µl of SybR green mix and 10.5 µl of cDNA mix per well (total volume

= 25 µl) in the appropriate multiwell plates. Dispense the SybR Green mix before the

cDNA mix. You can use the same filter tip as long as you pipet the same solution.

Cover the plate with appropriate optical adhesive lid.

4. Centrifuge the PCR plate at 4000 rpm for 5 min to get rid of any bubbles in the

samples.

5. Run the PCR following the Users Manual.

6. IMPORTANT: Before using the Bio-Rad machine the first time, you must be

instructed by an experienced user (currently: Jean-Denis or Dimitri).

VI. Analysis of Q-PCR results

1. Use the auto Ct determination mode. Manual setting of the Ct determination threshold

does not normally significantly alter the results. The regression method works nice

and is mathematic, it does not required your personal appreciation.

2. Determine relative expression levels using the -Ct method on an excel sheet or the

software of the Bio-Rad PCR machine. Use one sample as the reference to normalize

all other samples for the transcripts of interest.

3. Normalize your results in a way that the average of the transcript levels for the control

group is set to 100%.

 5

4. For gene expression analysis in limb buds, comparing groups of 9 limb bud pairs

is sufficient to detect significant differences using the two-tailed unpaired t-test. t-tests

require a normal distribution of the values in each of the groups (= different

genotypes).

For the statistical analysis use the PRISM software available in Christofori’s lab.

5. If required, Q-PCR results can be pooled from several plates by normalizing them.

IMPORTANT: For details, please discuss with an experienced user or consult the

appropriate manuals and statistical handbooks. You must understand how to analyze

your data correctly and detect variation due to experimental error. All results must be

statistically significant. No samples can be excluded, i.e. there are no outriggers.

VII. Primer design

1. Design specific primers using the Primer Express software.

2. The basic rules: the amplicon must span an intron/exon junction to avoid genomic

DNA amplification even if your RNA is DNase treated. Only if this is not possible,

then design primers within one exon and strictly use them on DNase-treated RNA

samples (see protocol III). Use default parameters in the RT-PCR mode with the

exception of the amplicon size that you have to set to 70 -150 bp. Primer pairs with a

score below 40 often work nicely. If scores are too high or the software finds no or

few pairs of primers, go back to the parameters and increase the window of acceptable

length of primers: 18 to 25 with an optimal length of 22. This often increases the

number of acceptable primer pairs. The default settings find primers for an annealing

temperature of 60°C, which is the annealing temperature used for Q-PCR.

 6

3. Run an in silico PCR of the UCSC genome browser website. You can interrogate

genomic and cDNA databases. The default settings are a minimal match of 15

nucleotides for each primer. Check that these setting are fitting with your primer

length (it corresponds to 7 mismatches in a 22 nucleotide primer). This allows you to

check that your primers are specific for the cDNA of interest and do not amplify

genomic DNA or other cDNAs. The perfect test is to verify the absence of an

amplicon in loss of function tissues.

4. Order the primers you have selected: We recommend to order 250 nmole dehydrated.

5. Upon arrival, resuspend the dehydrated primers in Elution Buffer (10mM TrisHCl

pH8.5) at 100µM. This is the stock to be stored at -80ºC in 100µl aliquots.

6. Prepare the working solution: 7.5µl primer A + 7.5µl primer B + 185µl Elution

Buffer. These working solutions are stored in aliquots at -20ºC. Use one aliquot per

experiment, do not refreeze.

7. Test the primer pair: on a standard cDNA preparation using the following dilution

series 1/ 0.1/ 0.01/ 0.001µl. Run a Q-PCR with the new primers and the normalizing

primers (that have an efficiency of 100%). It is important that the new primers and the

normalizers have a similar efficiency range. For example, the normalizer primers for

RPL19 are measured around cycle 20. Thus this range should be covered in the

dilution series for your new primers. For your information: a 10-fold dilution of a

particular cDNA corresponds to a delay of 3.33 cycles if the a reaction efficiency is

100%. The ratio between the normalizer and the cDNA of interest at different

dilutions should be a constant (with acceptable variation) and fall within the

concentration range used for your studies with other established primer pairs. The

melting curve must show a single peak, which indicates that there is only one major
 7

amplicon. You can also load your Q-PCR products on a high percentage agarose gel to

visualize them for control purposes.

8. Compare the amplification profile of a retrotranscribed and a non-retrotranscribed

RNA preparation at similar concentrations. No amplification should be detected in the

non-retrotranscribed sample treated with DNase. In case of non-specific

amplification, an about 10 cycle delay in comparison to specific amplification

indicates that the non-specific signal represents less than 0.1% of the total signal,

which is acceptable.

9. Please discuss all these matters with experienced users to make sure that you

evaluate your results correctly and according to lab standards.