Gene Expression analysis by qPCR requires a series of techniques, each carefully executed in order to generate a valid result. Because mRNA is inherently unstable, absolute care is needed to ensure that the transcripts have not degraded during extraction and subsequent manipulation. qPCR analysis for gene expression is also sometimes called RT-qPCR since reverse transcription generates the cDNA for amplification. It has been observed that reverse transcription introduces most of the variability in mRNA measurement.
Properties of the reverse transcription reaction in mRNA quantification. Ståhlberg, A., et. al. Clin. Chem. 2004a. 50(3), 509–515.
Once the mRNA has been reverse transcribed into cDNA, several different methods are commonly used to quantify the cDNA, termed absolute and relative quantification. Regardless of the method, a standard template of known concentration is needed to represent the target and validate assay performance. This standard can be a synthetic amplicon, a linearized plasmid, or even a PCR product purified from a previous reaction.
Figure 2: a four-fold dilution series of a target standard reveals 2-cycle intervals between CT values, or 100% efficient amplification.
Dilute this standard across a large range of copy number, with each dilution providing a starting point for amplification. Because these quantities are known in advance, the amplification of the series reveals the assay efficiency and sensitivity. Samples of unknown copy number can only be quantified should they fall in the bounds of this validation (within the range of the dilution series).
Amplification efficiency is indicated by the interval of CT values between dilutions. A 10-fold dilution series with intervals of 3.3 cycles indicates 100% efficiency, as determined by the formula: E = 10^(1/k) - 1. k is the dilution interval, also represented by the negative slope of the standard curve.