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

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, its practical applications are unexploited primarily because of the absence of an 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. (C) 2011 Elsevier Inc. All rights reserved.