Characterization of the 3’: 5’ ratio for reliable determination of RNA quality

Abstract

Determination of RNA quality is a critical first step in obtaining meaningful gene expression data. The PCR-based 3’: 5’ assay is an RNA quality assessment tool. This assay is a simple, fast and low-cost method of selecting samples for further analysis. However, the practical application is unexploited primarily because of the absence of a experimental threshold. We show that, by anchoring the 5’assay a specific distance from the 3’ end of the sequence and by spacing the 3’ at a distance of a number of nucleotides, a cutoff determines whether a sample is suitable for downstream quantification studies.