Inter-Laboratory Variability in Array-Based RNA Quantification Methods.

Abstract

Ribonucleic acids (RNA) are hypothesized to have preceded their derivatives, deoxyribonucleic acids (DNA), as the molecular media of genetic information when life emerged on earth. Molecular biologists are accustomed to the dramatic effects a subtle variation in the ribose moiety composition between RNA and DNA can have on the stability of these molecules. While DNA is very stable after extraction from biological samples and subsequent treatment, RNA is notoriously labile. The short half-life property, inherent to RNA, benefits cells that do not need to express their entire repertoire of proteins. The cellular machinery turns off the production of a given protein by shutting down the transcription of its cognate coding gene and by either actively degrading the remaining mRNA or allowing it to decay on its own. The steady-state level of each mRNA in a given cell varies continuously and is specified by changing kinetics of synthesis and degradation. Because it is technically possible to simultaneously measure thousands of nucleic acid molecules, these quantities have been studied by the life sciences community to investigate a range of biological problems. Since the RNA abundance can change according to a wide range of perturbations, this makes it the molecule of choice for exploring biological systems; its instability, on the other hand, could be an underestimated source of technical variability. We found that a large fraction of the RNA abundance originally present in the biological system prior to extraction was masked by the RNA labeling and measurement procedure. The method used to extract RNA molecules from cells and to label them prior to hybridization operations on DNA arrays affects the original distribution of RNA. Only if RNA measurements are performed according to the same procedure can biological information be inferred from the assay read out.