Gene Expression Analysis Using Quantitative Reverse Transcription-Polymerase Chain Reaction and a Multispecific Internal Control

Quantitation of very small amounts of specific messenger ribonucleic acids (mRNAs) extracted from tissues or cell cultures is a difficult and time-consuming task that necessitates the use of carefully controlled amplification procedures. Reverse transcription (RT) of the mRNA to cDNA followed by amplification using the polymerization chain reaction (RT-PCR) is widely used for simple detection purposes, but the procedure can be employed for accurate quantitation provided an appropriate internal control is present throughout the process. The choice of an internal control depends on the number of mRNAs that are to be quantitated, but it is essential that it incorporates exactly the same PCR priming sites as those chosen in the target mRNA. The amplification efficiency of the PCR is rarely 100% and often far lower and it is the primer pair that contributes the most towards PCR efficiency. In addition, because the RT step is also well under 100% efficient, it is highly desirable that the control be in the form of an RNA and that it be added to the total RNA before RT.