Mutant of Moloney murine leukemia virus reverse transcriptase exhibits higher resistance to common RT-qPCR inhibitors

Arezi, Bahram; McCarthy, Melissa; Hogrefe, Holly H.

doi:10.1016/j.ab.2010.01.024

Cited by 22 publications

(24 citation statements)

References 10 publications

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“…Sorting cells using TaqMan RT-PCR as an assay readout has never before been demonstrated, due both to the difficulty of preparing and handling stable, single-cell containing droplets and also lysate-mediated inhibition of the reaction in subnanoliter volumes (7,19,20). The technical advance key to enabling PACS has been our development of a robust microfluidic workflow that maintains compartmentalization of single-cell lysates at all times while also overcoming mammalian cell lysate-mediated inhibition of PCR (8).…”

Section: Resultsmentioning

confidence: 99%

Identification and genetic analysis of cancer cells with PCR-activated cell sorting

Eastburn

Sciambi

Abate

2014

Nucleic Acids Research

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Cell sorting is a central tool in life science research for analyzing cellular heterogeneity or enriching rare cells out of large populations. Although methods like FACS and FISH-FC can characterize and isolate cells from heterogeneous populations, they are limited by their reliance on antibodies, or the requirement to chemically fix cells. We introduce a new cell sorting technology that robustly sorts based on sequence-specific analysis of cellular nucleic acids. Our approach, PCR-activated cell sorting (PACS), uses TaqMan PCR to detect nucleic acids within single cells and trigger their sorting. With this method, we identified and sorted prostate cancer cells from a heterogeneous population by performing >132 000 simultaneous single-cell TaqMan RT-PCR reactions targeting vimentin mRNA. Following vimentin-positive droplet sorting and downstream analysis of recovered nucleic acids, we found that cancer-specific genomes and transcripts were significantly enriched. Additionally, we demonstrate that PACS can be used to sort and enrich cells via TaqMan PCR reactions targeting single-copy genomic DNA. PACS provides a general new technical capability that expands the application space of cell sorting by enabling sorting based on cellular information not amenable to existing approaches.

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Section: Resultsmentioning

confidence: 99%

Identification and genetic analysis of cancer cells with PCR-activated cell sorting

Eastburn

Sciambi

Abate

2014

Nucleic Acids Research

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“…Whether glycerol and trehalose can further increase the thermal stabilities of our triple and quadruple variants will be the subject of our next investigations. In regard of this, it was recently reported that the stable MMLV RT variant, E69K/E302R/W313F/L435G/N454K, is highly resistant to common RT-PCR inhibitors including guanidine, thiocyanate, ethanol, formamide, ethylenediaminetetraacetic acid (EDTA), and plant-related acidic polysaccharides (Arezi et al, 2010).…”

Section: Cdna Synthesis By the Highly Stable Multiple Variantsmentioning

confidence: 99%

Increase in thermal stability of Moloney murine leukaemia virus reverse transcriptase by site-directed mutagenesis

Yasukawa

Mizuno

Konishi

et al. 2010

Journal of Biotechnology

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We hypothesized that the thermal stability of Moloney murine leukaemia virus reverse transcriptase (MMLV RT) will increase with increases in its ability to bind with a template-primer (T/P). To test this hypothesis, we introduced positive charges into MMLV RT by site-directed mutagenesis at positions that have been implicated in the interaction with T/P. Thirty-six variants were constructed in which one of the twelve residues (Glu69, Gln84, Asp108, Asp114, Glu117, Glu123, Asp124, Glu286, Glu302, Trp313, Leu435, and Asn454) was replaced with Lys, Arg, or Ala, and these were expressed in Escherichia coli. In about half of these 36 variants, thermal inactivation at 50°C was reduced in the presence of the T/P, which suggested that this strategy was effective at stabilizing MMLV RT. We next combined three of the 36 mutations, Glu286→Ala, Glu302→Lys, and Leu435→Arg, and the mutation, Asp524→Ala, which is known to abolish the RNase H activity and increase the stability. Temperatures of 54 and 56°C reduced the initial reverse transcription activity by 50% over a 10-min incubation in the triple variant E286R/E302K/L435R and quadruple variant E286R/E302K/L435R/D524A, respectively. These temperatures were higher than that observed for WT (45°C). The highest temperature at which the triple and quadruple variants exhibited cDNA synthesis activity was 60°C, which was again higher than for WT (54°C). Thus, highly stable MMLV RT variants were generated by this mutation strategy.

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“…For RNA detection [4], the most fundamental step is the in-vitro reverse transcription (RT) of RNA to its complementary DNA (cDNA), catalyzed by the enzyme reverse transcriptase [5], [6], [7]. Many factors such as the quality of the RNA, the presence of inhibitors co-extracted with the RNA, as well as the RT efficiency are known to affect the total yield of cDNA [8], [9].…”

Section: Introductionmentioning

confidence: 99%

Quantitative Assessment of the Sensitivity of Various Commercial Reverse Transcriptases Based on Armored HIV RNA

et al. 2010

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BackgroundThe 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 FindingsWe 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/SignificanceWe 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.

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Mutant of Moloney murine leukemia virus reverse transcriptase exhibits higher resistance to common RT-qPCR inhibitors

Cited by 22 publications

References 10 publications

Identification and genetic analysis of cancer cells with PCR-activated cell sorting

Identification and genetic analysis of cancer cells with PCR-activated cell sorting

Increase in thermal stability of Moloney murine leukaemia virus reverse transcriptase by site-directed mutagenesis

Quantitative Assessment of the Sensitivity of Various Commercial Reverse Transcriptases Based on Armored HIV RNA

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