Quantitating mRNAs with Relative and Competitive RT-PCR

Reverse transcription coupled with the polymerase chain reaction (RT-PCR), permits amplification of cellular RNA. Gene expression can be measured even when the message copy number is very low (1–10 copies per cell) (1 ) or the sample size is very small (1–1000 cells) (2 ,3 ). In RT-PCR, an RNA sample is primed with gene-specific primers, oligo dT, or random primers, and copied into a complementary DNA sequence, or cDNA, using a retroviral reverse transcriptase. The first-strand cDNA from the RT amplification of cellular RNA. Gene expression can be measured even when the message copy number is very low (1–10 copies per cell) (1 ) or the sample size is very small (1–1000 cells) (2 ,3 ). In RT-PCR, an RNA sample is primed with gene-specific primers, oligo dT, or random primers, and copied into a complementary DNA sequence, or cDNA, using a retroviral reverse transcriptase. The first-strand cDNA from the RT reaction is subsequently amplified using gene-specific primers and a thermostable DNA polymerase (e.g., Taq polymerase).