Quantitative PCR is a method used to detect relative or absolute gene expression level. All qPCR involves the use of fluorescence to detect the threshold cycle (Ct) during PCR when the level of fluorescence gives signal over the background and is in the linear portion of the amplified curve. This Ct value is responsible for the accurate quantization of qPCR.
SYBR Green is a dye that intercalates with double-stranded DNA. This intercalation causes the SYBR to fluoresce. The qPCR machine detects the fluorescence and software calculates Ct values from the intensity of the fluorescence.
This protocol will cover the SYBR Green quantitative PCR technique, and includes suggestions about which kits to use and an overview of how to analyze your data.
Quantitative PCR was initially developed to detect the copy number of transcribed mRNA, and that is basically what we still do when we perform qPCR.
1.) First, RNA must be isolated from your samples. Use a technique for isolating RNA that suits you best. TRIzol methods work well, and there are many good kits for it available.
Recommended: For isolating from cell culture and mouse liver, I use Qiagen’s RNeasy Mini Kit (Cat. No. 74104)
This kit will work on many tissues and cell types. For aorta, I use Qiagen’s RNeasy Fibrous Tissue Mini Kit (Cat. No. 74704)
2.) In order to preserve RNA samples, which are very vulnerable to degradation at room temperature, I recommend using a first-strand DNA synthesis on your RNA in preparation for qPCR, as opposed to running RT-PCR simultaneously with qPCR.
Recommended: For first-strand DNA syntheses, I use Invitrogen’s SuperScript III First-Strand Synthesis System for RT-PCR (Cat. No. 18080-051)
This will create cDNA in a 1:1 ratio to the RNA in your sample.
3.) For the qPCR itself, you will need your cDNA samples, standards made from the samples, primers specific for your genes of interest, and a SYBR Green mix (which will include SYBR Green dye, Taq Polymerase, ROX, and dNTP all in one).
Recommended: SYBR Green kits are available from many companies. I recommend Qiagen’s QuantiTect SYBR Green PCR Kit (Cat. No. 204143 for 500 rxns, Cat. No. 204145 for 2500 rxns)
You will also need an internal control as a point of comparison for your data. Choose a housekeeping gene that is endogenously expressed in your cell type (e.g. β-2 microglobulin, β-actin).
4.) Finally, before making the plate, make sure to sign up to use a quantitative PCR machine ahead of time. Two available options:
1. You can sign up to use the one in the Genotyping Core on 5th floor Gonda by calling x72461. The cost is $40 per plate, and you need to sign in when you submit the plate.
2. Or you can use the one in Dr. Steve Young’s laboratory. Sign up online at http://calendar.yahoo.com with login “younglabqpcr” and password “7500abi”.
Every gene you run on qPCR will need to be run with a standard curve in order to relatively quantitate the Ct values of your samples.
The following protocol assumes that you have created cDNA from your RNA prior to qPCR. If you followed the Invitrogen SuperScript III kit’s protocol, you should start with 21μl of cDNA per sample.
1.) Dilute each cDNA sample ~4-fold. In this case, dilute your 21μl with 59μl H2O for a final volume of 80μl. Vortex and spin down.
2.) Pool an equal amount from each sample into a single tube. This will be Standard 1, your high standard. To determine how much to pull from each sample, calculate how much you will have left in each sample and what the final volume of your standards will be. (This is to have approximately the same final volume of standards and samples.)
Ø Example.)
Take 30μl from each of 12 samples and pool for Standard 1 with a final volume of 360μl.
Final sample volume will be sample volume * 5 after a five-fold dilution (see below). In this case, (80μl - 30μl) * 5 = 250μl of each sample, final volume.
Take 90μl of Standard 1 in a new tube labeled Standard 2. Dilute Standard 2 with 270μl H2O for a final volume of 360μl. Repeat up to Standard 5.
3.) Create the rest of your standards by taking out 1/4th of the last standard and diluting it 4-fold. Each standard will have a value assigned to it, as below.
Example.)
Standard Number | Dilution Factor | Dilutions | Value |
Standard 1 | Pool | 360μl Standard 1 | 25600 |
Standard 2 | 1:4 | 90μl Stnd. 1 + 270μl H2O | 6400 |
Standard 3 | 1:16 | 90μl Stnd. 2 + 270μl H2O | 1600 |
Standard 4 | 1:64 | 90μl Stnd. 3 + 270μl H2O | 400 |
Standard 5 | 1:256 | 90μl Stnd. 4 + 270μl H2O | 100 |
4.) Remember to vortex and spin down after each dilution step!
Dilute your samples (cDNA) further in a 1:5 dilution with H2O. For example, dilute your remaining 50μl of sample in 200μl H2O for a final volume of 250μl.
Before making the plate, draw a layout of how you will pipette it ahead of time so you know what is in each well.
Use plates appropriate to the machine you’ll be using. For the ABI7500 Fast PCR system, use Optical 96-Well Fast Thermal Cycling Plates from ABI (Part No.: 4346906). Use Optical Caps from ABI (Part No.: 4323032) with this plate.
Example) Two genes run on one plate. 18 samples total.