Quantitative Real-Time PCR

Overview

The following protocol Real-time or qPCR is generally used to compare cDNA levels of one sample to a second sample. Real-time PCR uses either a generic dye that binds non-specifically to dsDNA (sybergreen) or a specific primer probe (Taqman) designed to anneal to the center of the PCR product that will fluoresce only after product is made. The real-time PCR machine measures fluorescence after each thermo cycle. If done correctly, the PCR reaction causes an exponential increase in the amount of dsDNA, resulting in an S shaped curve of fluorescence.

The amount of product can be compared to other samples by comparing the cycle number where the exponential increase began. This cycle number is called the "cycle threshold" or Ct value. Generally, a shift down 1 cycle corresponds to roughly 2-3 times more starting cDNA. Determining the Cycle Threshold is generally done by graphing the 2nd derivative of the fluorescence curve, which gives you a peak where exponential increase in fluorescence is at a maximum. Otherwise you can manually set a baseline by eyeballing all the graphs, and the point where the curves cross the baseline becomes the Ct value.

Controls: Because real-time PCR requires comparisons, you need to have a "housekeeping" gene whose expression does not change between your conditions. This is not trivial. Genes frequently used include HPRT, Gapdh or beta-actin. However, mRNA levels of these genes are known to fluctuate in some cell types after certain treatments. Ideally, if you have gene array data comparing the two conditions, you can identify mRNA transcripts that do not change between conditions, and then use that transcript as your control.

Primers should be designed to cross intron boundaries and the product should be around 100 bp. This minimizes the chance of contaminating product from remaining genomic DNA and increases the efficiency of the reaction.

The efficiency of the reaction also needs to be accounted for both the housekeeping gene and the gene of interest. To calculate efficiency, a series of different starting amounts of DNA are run,

DNA concentrations used to calculate PCR efficiency

  • To calculate Efficiency graph the Log of DNA input on x axis (assume 1 µl cDNA = 1 µg DNA) against the Ct value on the y axis
  • Calculate the linear curve fit to generate a slope.
  • Efficiency = 10^(-1/slope) 2.0 = 100% efficient
  • acceptable range is from 1.8 to 2.2 (90 to 110%)

Melt temperature: Graphing the melt temperature should result in a single sharp peak, indicating one value. The melt temp should also be somewhat equivalent between the two samples.

Inflection of fluorescence curve should be dramatic. 2nd derivative peak should be sharp.

Protocol

  • Keep all ingredients on ice
  • All samples should be run in triplicate. Make a map of all reactions
  • Make cocktails for each triplicate. For example, make a cocktail of 80 µl for three triplicates of 25 µl each. Add enough DNA for three samples to the aliquot, and then aliquot into three wells or tubes. This will minimize pipetting error. Try to make everything in batches to avoid introducing pipetting error.

PCR reaction using 2X QuantiTect Sybr Green PCR Master Mix from Roche

PCR reaction using 2X Faststart Universal Sybr Green Master Mix from Roche (ala Juan Abrahante)

  • Aliquot samples into PCR tubes or plates
  • Run qPCR machine using the following program: 95°C 2 min. 40 cycles of 94°C 15 sec, 55°C 30 sec, 72°C 30 sec. Final anneal 72°C 1 min. Melt Curve from 55°C to 95°C for 20 min.

Operating the Applied Biosystems 7300 Real Time PCR system

  • Aliquot samples into sample plates (ABI qRT-PCR, USA scientific Temp Plate, semi-skirt 0.2 mL, natural Ustores Cat # CX13506).
  • Cover plate with adhesive film (Micro Amp optical adhesive film, PCR compatible DNA/RNA/RNase free, Applied Biosystems Cat # 4311971).
  • Open drawer for sample loading-push in with hands, drawer will open automatically.
  • Place 9600 black tray in loading dock (for use with 96 well plates only) then place your 96 well plate in the black tray.
  • Push drawer gently once sample plate loaded. Drawer will close automatically.
  • Turn on 7300 Real Time PCR machine using grey power button on right side
  • On the computer, double click the 7300 system software to open
  • Once software is open, click “create new document” to begin analysis
  • In the Define Document window, set “Assay” field to “Standard Curve (Absolute Quantification)”, and leave everything else as is.
  • Click on the “Browse” button, browse to your template file, highlight the filename, then click open. If you don't already have a template file defined, read the help documents to set one up.
  • Adjust the template to your experiment. Select wells of interest, hit command I or open Well Inspector on the setup map. Enter the sample name. Select SYBR (but not ROX). In the "Passive Reference" pull down window in the lower right screen select ROX. After entering all samples, save the document.
  • Click on the instrument tab to set up the program. A standard program is 95ºC 10 min, 40 cycles of 95ºC for 15 sec and 60ºC for 45 sec, followed by a melt curve of 95ºC for 15 sec followed by 60ºC for 1 min follwed by 95ºC for 15 sec.
  • Save the file as an SDS Document
  • Return to the Instrument window and hit start.
  • Most programs take ~2.5 hours to run.
  • Return to machine and click "OK" in the screen showing that the run has completed.
  • Click on the "Report" tab, select file > export > results. The program will create a spreadsheet with the results.
  • Save the results spreadsheet to a USB storage device.
  • Open the plate drawer and remove samples. These can be run on a gel to check band size.
  • Close drawer by pushing in gently
  • Turn off machine by pressing the green power button on the right side.

Data Analysis

This procedure is known as delta-delta Ct. A newer method, outlined by Pfaffl (Nucleic Acids Research, 2001), is supposedly better. So I will be using that one from now on. To calculate fold change between a baseline and a condition use the following formula. Note: this formula is considered a delta-delta Ct calculation

Fold Change = (P ^ (Ct(x) - Ct(y))) / (H ^ (Ct(a) - Ct(b)))

Note: the above equation works if mRNA levels increase (Ct value goes down). If mRNA levels decrease (Ct values rise), the formula is -1 / the formula above. If you have not calculated the efficiency of your primers, just set P and H equal to 2.

  • P = calculated efficiency of the gene of interest qPCR
  • H = calculated efficiency of the housekeeping qPCR
  • Ct(x) = Cycle threshold of gene of interest at "baseline"
  • Ct(y) = Cycle threshold of gene of interest after treatment/condition
  • Ct(a) = Cycle threshold of housekeeping gene at "baseline"
  • Ct(b) = Cycle threshold of housekeeping gene after treatment/condition