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J Biomol Tech. 2010 September; 21(3 Suppl): S45.
PMCID: PMC2918151

RT-qPCR Analysis of Degraded RNA using Five Different Pre-Amplification Methods

M.L. Shane,2 M. Kohlmeyer,2 and T. Hunter2
1Microarray Core Lab, University of Vermont, Burlington, VT, United States;
2DNA Analysis Facility, University of Vermont, Burlington, VT, United States

Abstract

RP-77

As the analysis of degraded RNA continues to be contemplated in RT-qPCR studies, the use of pre-amplification techniques might help improve data quality in special circumstances. The focus of this study was to evaluate the effectiveness of five different pre-amplification strategies on four different levels of RNA degradation. Ambion's First Choice Brain Reference RNA was non-chemically degraded to RIN values of 9, 6, 4, and 2 as determined by the Agilent Bioanalyzer.Each RNA was subjected to pre-amplification using standard Eberwine techniques, ExpressArt by AmpTec, NuGEN's WT Ovation, WT Pico, and WT FFPE kits and compared with the matching first strand cDNA preparation for B-actin at 207 bp, B-actin at 695 bp, and GAPDH at 1057 bp (positon from 3' end). Results indicated that amplification using a combined oligo-d[T] and randomers were less affected by degradation and CT values were consistent for all levels of degradation when the assay location was greatest from the 3' end of the transcript. Samples that were not pre-amplified or amplified using only oligo-d[T] showed a steady climb in CT value as degradation increased regardless of location.


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