Variability of the Reverse Transcription Step: Practical Implications

Bustin, Stephen A.; Dhillon, Harvinder S.; Kirvell, Sara; Greenwood, Christina; Parker, Michael; Shipley, Gregory L.; Nolan, Tania

doi:10.1373/clinchem.2014.230615

Cited by 73 publications

(61 citation statements)

References 39 publications

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“…The experiments here suggest that qRT-PCR has lower intra- and inter-assay variation compared to QT-NASBA. The higher precision of qRT-PCR is evident for the entire range of gametocyte densities used in the dilution series, despite involving extra reaction steps, such as DNase treatment and cDNA production, which are known to be a source of variation [27]. These two steps were performed once for each dilution series density, for the three different NF54 cultures and the NF135 and the NF166 cultures.…”

Section: Discussionmentioning

confidence: 99%

Comparison of molecular quantification of Plasmodium falciparum gametocytes by Pfs25 qRT-PCR and QT-NASBA in relation to mosquito infectivity

Pett

Gonçalves

Dicko

et al. 2016

Malar J

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BackgroundQuantifying gametocyte densities in natural malaria infections is important to estimate malaria transmission potential. Two molecular methods (Pfs25 mRNA quantitative reverse transcriptase PCR (qRT-PCR) and Pfs25 mRNA quantitative nucleic acid sequence based amplification (QT-NASBA)) are commonly used to determine gametocyte densities in clinical and epidemiological studies and allow gametocyte detection at densities below the microscopic threshold for detection. Here, reproducibility of these measurements and the association between estimated gametocyte densities and mosquito infection rates were compared.MethodsTo quantify intra- and inter-assay variation of QT-NASBA and qRT-PCR, a series of experiments was performed using culture-derived mature Plasmodium falciparum gametocytes from three different parasite isolates (NF54, NF135, NF166). Pfs25 mRNA levels were also determined in samples from clinical trials in Mali and Burkina Faso using both methods. Agreement between the two methods and association with mosquito infection rates in membrane feeding assays were assessed.ResultsIntra- and inter-assay variability was larger in QT-NASBA compared to qRT-PCR, particularly at low gametocyte densities (< 1 gametocyte per μL). Logistic models, including log-transformed gametocytaemia estimated by QT-NASBA, explained variability in mosquito feeding experiment results as well as log-transformed gametocytaemia by qRT-PCR (marginal R2 0.28 and 0.22, respectively). Densities determined by both methods strongly correlated with mosquito infection rates [Spearman’s rank correlation coefficient, 0.59 for qRT-PCR and 0.64 for QT-NASBA (P < 0.001 for both)]. Gametocyte densities estimated by qRT-PCR were higher than levels estimated by QT-NASBA or light microscopy at high densities (>100 gametocyte per μL). Samples collected in one of the two transmission studies had extremely low gametocyte densities by both molecular methods, which is suggestive of RNA degradation due to an unknown number of freeze–thaw cycles and illustrates the reliance of molecular gametocyte diagnostics on a reliable cold-chain.ConclusionsThe experiments indicate that both qRT-PCR and QT-NASBA are of value for quantifying mature gametocytes in samples collected in field studies. For both assays, estimated gametocyte densities correlated well with mosquito infection rates. QT-NASBA is less reproducible than qRT-PCR, particularly for low gametocyte densities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1584-z) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Comparison of molecular quantification of Plasmodium falciparum gametocytes by Pfs25 qRT-PCR and QT-NASBA in relation to mosquito infectivity

Pett

Gonçalves

Dicko

et al. 2016

Malar J

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“…It has been shown that the results of the RT depended on the characteristics of the RT enzyme, samples, RNA concentrations and assays [88]. …”

Section: Normalization Quality Control and Reportingmentioning

confidence: 99%

qPCR-Based Methods for Expression Analysis of miRNAs

et al. 2019

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Several approaches for miRNA expression analysis have been developed in recent years. In this article, we provide an updated and comprehensive review of available qPCR-based methods for miRNA expression analysis and discuss their advantages and disadvantages. Existing techniques involve the use of stem–loop reverse transcriptase–PCR, polyadenylation of RNAs, ligation of adapters or RT with complex primers, using universal or miRNA-specific qPCR primers and/or probes. Many of these methods are oriented towards the expression analysis of mature miRNAs and few are designed for the study of pre-miRNAs and pri-miRNAs. We also discuss findings from articles that compare results from existing methods. Finally, we suggest key points for the improvement of available techniques and for the future development of additional methods.

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“…Yet another paper reported gene-related factors as the primary determinants of RT variability and called for an evaluation of the RT robustness of control genes in RT-qPCR normalisation [40]. A recent paper has demonstrated that the fold changes reported by most RNA biomarkers are well within the range of variability observed when multiple RT reactions are carried out on the same template [41]. The conclusions from these papers are rather stark and place question marks behind many of the results reported not just in the biomedical literature but in the scientific literature as a whole.…”

Section: Technical Issuesmentioning

confidence: 99%