Detection limits of several commercial reverse transcriptase enzymes: impact on the low- and high-abundance transcript levels assessed by quantitative RT-PCR

Lévesque-Sergerie, Jean-Philippe; Duquette, Mathieu; Thibault, C.; Delbecchi, Louis; Bissonnette, Nathalie

doi:10.1186/1471-2199-8-93

Cited by 78 publications

(91 citation statements)

References 31 publications

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“…At this regards, in this study an external in vitro synthesized cRNA standard is generated in order to obtain a calibration standard curve, thereby offering some advantages as control and standardization of RNA extraction and reverse transcription. The sensitivity of our assay was found to be of 100 copy/reaction with a linearity range from 10 (9). Overall, the unique condicio sine qua non for cRNA standard synthesis is represented by the presence of a RNA polymerase promoter-binding site into the vector containing the amplicon of interest (many commercial products, comprising those used in our protocol, are characterized by the presence of T7-RNA polymerase promoter) and the choice of the in vitro transcription kit depends on it (5).…”

Section: Discussionmentioning

confidence: 99%

Improvement of HRV Quantification Using cRNA-Based Standards for Real Time RT-PCR

et al. 2010

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Real Time RT-PCR developed in recent years represents an useful tool in the diagnosis of RNA viruses. In order to accurately quantify and normalize a RNA target, efficiency of reverse-transcription must be considered. In this study a cRNA-standard based quantitative Real Time RT-PCR have been developed for HRV quantification on bronchoalveolar lavage (BAL) specimens. Results has been compared to a quantitative plasmid standard-based Real Time RT-PCR previously developed by us.Large amount of pHRV was linearized and purified. Blunt ends were generated and cRNA production was carried out. Dilutions of cRNA were generated and dynamic range, intraand inter-test variability, sensitivity and limit of detection were evaluated. Sixty-seven BAL, previously resulted positive to our plasmid standard-based method, were evaluated using cRNA-standard quantification. cRNA curve showed a broad dynamic range with a good intra-and inter-test variability, with an average of 3.23 threshold cycles more in comparison to plasmid standard-based curve. In terms of specimen quantification, a difference of 1.07log was found, showing a significant underrate using plasmid standard-based quantification.The method for cRNA-standard construction seems more suitable for quantification of RNA viruses, in order to normalize the quantification in reverse-transcription.

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Section: Discussionmentioning

confidence: 99%

Improvement of HRV Quantification Using cRNA-Based Standards for Real Time RT-PCR

et al. 2010

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“…The formula takes into account the respective amplicon length generated during real-time PCR (Supplementary Table 3) as described (Lalancette et al 2008b). Only the absolute transcript copies were normalized using the exogenous EGFP transcripts as described (Levesque-Sergerie et al 2007), whereas the relative method readily includes a normalization using PPIA, an endogenous 'housekeeping' gene. All points of the standard curves and each query samples were carried out with triplicates and run in the ABI PRISM 7500 Sequence Detection System with software version 1.4 (Applied Biosystems).…”

Section: Qrt-pcr Data Analysismentioning

confidence: 99%

“…In order to normalize the difference in RT efficiencies across samples, each RT reactions was spiked EGFP mRNA. An equivalent of 1 pg/mg of spermatozoal RNA was added in pooled RNA and each RT reaction made with 1 mg included an equal amount of EGFP transcripts in each 20 ml reaction as described (Levesque-Sergerie et al 2007). …”

Section: Reverse Transcriptionmentioning

confidence: 99%

Spermatozoal transcriptome profiling for bull sperm motility: a potential tool to evaluate semen quality

Bissonnette¹,

Lévesque-Sergerie²,

Thibault³

et al. 2009

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Regarding bull fertility, establishing an association between in vitro findings and field fertility requires a multi-parametric approach that measures the integrity of various structures and dynamic functions, such as motion characteristics, among others. The heterogeneous RNA pattern of spermatozoa could be used in genomic analysis for evaluating both spermatogenesis and fertility potential of semen, mainly because of the static status of the transcriptome of this particular differentiated cell. In a previous study, we determined that some spermatozoal transcripts identified by PCR-based cDNA subtraction are associated with non-return rate, a field fertility index. In the present study, the microarray technology was used in conjunction with differential RNA transcript extraction. We have shown that among these genes, some transcripts are also associated with the motility status of a population of sperm cells fractionated from the same ejaculate. We highlighted a systematic data analysis and validation scheme important for the identification of significant transcripts in this context. With such an approach, we found that transcripts encoding a serine/threonine testis-specific protein kinase (TSSK6) and a metalloproteinase non coding RNA (ADAM5P ) are associated with high-motility status (P!0.001), also confirmed by quantitative PCR (PZ0.0075). This association was found only when transcripts were extracted using the hot-TRIzol protocol, whereas the cold-TRIzol RNA extract comprised mitochondrial transcripts. These results demonstrate that some transcripts previously identified in association with field fertility are also found associated with in vitro motility provided that a stringent RNA extraction protocol is used.

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“…Increase of commercial master mix kits enabled diagnostic laboratories to implement real-time PCR. It has been demonstrated that the reverse transcription reaction mainly depends on the initial molecular step (the reverse transcription), choice of master mix reagents, quality of enzymes, concentration of reaction mix components, such as primers, probes and methodology used that can impact the sensitivity of results for real-time PCR assays (SERGERIE et al, 2007;BUZARD et al, Ciência Rural, v.46, n.9, set, 2016.…”

Section: Resultsmentioning

confidence: 99%

Canine distemper virus detection by different methods of One-Step RT-qPCR

et al. 2016

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Three commercial kits of One-Step RT-qPCR were evaluated for the molecular diagnosis of Canine Distemper Virus. Using the kit that showed better performance, two systems of Realtime RT-PCR (RT-qPCR) assays were tested and compared for analytical sensitivity to Canine Distemper RESUMO Três kits comerciais de One-Step RT-qPCR foram avaliados para o diagnóstico molecular do Vírus da Cinomose Canina.Utilizando o kit que apresentou melhor desempenho, dois sistemas de RT-PCR em tempo real (RT-qPCR) foram comparados quanto à sensibilidade analítica na detecção do RNA do Vírus da Cinomose Canina:One-Step RT-qPCR (Sistema A) e One-Step RT-qPCR seguido da NESTED-qPCR (Sistema B).Os limites de detecção dos dois sistemas foram determinados utilizando diluição

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Detection limits of several commercial reverse transcriptase enzymes: impact on the low- and high-abundance transcript levels assessed by quantitative RT-PCR

Cited by 78 publications

References 31 publications

Improvement of HRV Quantification Using cRNA-Based Standards for Real Time RT-PCR

Improvement of HRV Quantification Using cRNA-Based Standards for Real Time RT-PCR

Spermatozoal transcriptome profiling for bull sperm motility: a potential tool to evaluate semen quality

Canine distemper virus detection by different methods of One-Step RT-qPCR

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