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
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.
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)
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.