Quantitative assessment of the sensitivity of various commercial reverse transcriptases based on armored HIV RNA

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, especi...

Full description

Saved in:
Bibliographic Details
Published in:PloS one 2010-11, Vol.5 (11), p.e13931-e13931
Main Authors: Okello, John B A, Rodriguez, Linda, Poinar, Debi, Bos, Kirsten, Okwi, Andrew L, Bimenya, Gabriel S, Sewankambo, Nelson K, Henry, Kenneth R, Kuch, Melanie, Poinar, Hendrik N
Format: Article
Language:English
Subjects:
Cell Biology/Gene Expression
Comparative analysis
Complementary DNA
Copy number
Deoxyribonucleic acid
DNA
DNA polymerase
DNA polymerases
DNA, Complementary - biosynthesis
DNA, Complementary - genetics
Efficiency
Enzymes
Foot & mouth disease
Gene expression
Genetic transcription
Genetics and Genomics
genomics
Health sciences
HIV
HIV (Viruses)
HIV - genetics
Human immunodeficiency virus
Humans
Lysates
Molecular Biology
nucleic acids
Paraffin
Pathology
Polymerase chain reaction
Purification
Rankings
Reproducibility
Reproducibility of Results
Reverse Transcriptase Polymerase Chain Reaction - methods
Reverse transcription
Reverse Transcription - genetics
Ribonucleic acid
RNA
RNA, Viral - genetics
RNA, Viral - metabolism
RNA-directed DNA polymerase
RNA-Directed DNA Polymerase - metabolism
Sensitivity
Sensitivity analysis
Studies
Tissues
Trends
Virology/Diagnosis
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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. 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. 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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0013931