Benchmarking miRNA Expression Levels in Degraded RNA Samples Using Real-Time RT-qPCR and Microarray Technologies

摘要

The Nucleic Acid Research Group (NARG) has conducted experiments to determine the impact of RNA integrity and priming strategies on cDNA synthesis and Real-Time RT-qPCR. As a continuation of the RNA integrity theme, this year's study was expanded to evaluate the impact of RNA integrity on priming strategies for the analysis of nine miRNA targets using Real-Time RT-qPCR. The nine targets were selected based on data obtained by the Microarray Research Group (MARG) and represent groups of miRNAs that are expressed at low, medium, or high levels in the First Choice human brain reference RNA sample. The two RT-qPCR priming strategies tested in this study include the miRNA TaqMan assay (Megaplex) of ABI and the RT2 miRNA qPCR assay of Qiagen/SA Biosciences. The basis for the ABI assay design is a target specific stem-loop structure and reverse-transcription primer, while the Qiagen design combines poly (A) tailing and a universal reverse transcription in one cDNA synthesis reaction. For this study to assess both RT methods, samples that were used as templates were human brain reference RNA that has been subjected to controlled degradation using RNase A to RIN (RNA Integrity Number) values of 7 (good), 4 (moderately degraded), and 2 (severely degraded). In addition to this Real-Time RT-qPCR data, the same RNA templates were further analyzed using universal poly (A) tailing followed by hybridization to Affymetrix miRNA GeneChips. We present some insights into RT priming strategies for miRNA and contrasts qPCR results obtained using different technologies.