Assessment of RNA quality by semi-quantitative RT-PCR of multiple regions of a long ubiquitous mRNA

A simple method to assess the degree of degradation present in a total RNA preparation from cells or tissues is based on the increasing probability of RNA cleavage with increasing length of an RNA molecule. Under ideal condit ions, reverse transcription of a particular mRNA species with oligo-dT as t he primer generates a population of cDNAs, terminating at the 5' end of the mRNA if all template RNA molecules are intact, or at the first cleavage si te 5' to the polyA if some template RNAs are partially degraded. Consequent ly, for cellular RNA preparations with some degradation, the 5' end of an m RNA is represented in the cDNA population to a lesser extent than the 3' en d of the mRNA. We describe a sensitive assay of mRNA quality that compares the relative PCR amplification of 5' and 3' regions of a long and ubiquitou s mRNA following oligo dT-primed reverse transcription.