Quantitative Assessment of the Sensitivity of Various Commercial Reverse Transcriptases Based on Armored HIV RNA
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Date
2010
Journal Title
Journal ISSN
Volume Title
Publisher
PLoS ONE
Abstract
The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is
the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically
sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are
many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison
of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues.
Methodology/Principal Findings: We performed RT-qPCR to assess the sensitivity and reproducibility of 11 commercially
available reverse transcriptases in cDNA synthesis from low copy number RNA levels. As target RNA, we used a serially
known number of Armored HIV RNA molecules, and observed that 9 enzymes we tested were consistently sensitive to
,1,000 copies, seven of which were sensitive to ,100 copies, while only 5 were sensitive to ,10 RNA template copies
across all replicates tested. Despite their demonstrated sensitivity, these five best performing enzymes (Accuscript, HIV-RT,
M-MLV, Superscript III and Thermoscript) showed considerable variation in their reproducibility as well as their overall
amplification efficiency. Accuscript and Superscript III were the most sensitive and consistent within runs, with Accuscript
and Superscript II ranking as the most reproducible enzymes between assays.
Conclusions/Significance: We therefore recommend the use of Accuscript or Superscript III when dealing with low copy
number RNA levels, and suggest purification of the RT reactions prior to downstream applications (eg qPCR) to augment
detection. Although the results presented in this study were based on a viral RNA surrogate, and applied to nucleic acid
lysates derived from archival formalin-fixed paraffin embedded tissue, their relative performance on RNA obtained from
other tissue types may vary, and needs future evaluation.
Description
Keywords
Commercial Reverse Transcriptases, Armored HIV RNA
Citation
Okello JBA, Rodriguez L, Poinar D, Bos K, Okwi AL, et al. (2010) Quantitative Assessment of the Sensitivity of Various Commercial Reverse Transcriptases Based on Armored HIV RNA. PLoS ONE 5(11): e13931. doi:10.1371/journal.pone.0013931