Application of a master equation for quantitative mRNA analysis using qRT-PCR

The qRT-PCR has been widely accepted as the assay of choice for mRNA quantification. For conventional practice, housekeeping genes have been applied as internal reference for data normalization and analysis since the technology appeared. However, housekeeping genes vary under different conditions and environmental stimuli and no commonly accepted housekeeping gene references are available. Accurate data acquisition and data reproducibility remain challenging and it is difficult to compare results from different experimental sources. Using yeast and a Fusarium fungus as examples, we demonstrate the independent performance of a sole reference gene, CAB, designated as a constant manual threshold for data acquisition, normalization, and analysis for multiple plate reactions. A robust master equation based on the CAB reference and the set of calibration control genes thereafter was established to estimate mRNA abundance for the same RNA background reactions. A valid range of amplification efficiency between 95% and 100% was observed for the control genes in different RNA background applied on an ABI real time PCR 7500 system. This newly developed robust quality control system provides a reliable means for absolute quantification of mRNA using the qRT-PCR, simplifies the conventional qRT-PCR procedures, and increases data reliability, reproducibility, and throughput of the assay.