Talking the talk, but not walking the walk: RT-qPCR as a paradigm for the lack of reproducibility in molecular research

Bustin, Stephen A.; Nolan, Tania

doi:10.1111/eci.12801

Cited by 92 publications

(98 citation statements)

References 161 publications

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“…Given the significance of empirical validation, it is important that any publication include that essential information [8] , [9] , [10] , [11] . Several reports have been published recently that together scored thousands of peer-reviewed papers in a wide collection of journals ranging from low to high impact factors [12] , [13] , [14] , [15] , [16] , [17] . All concluded that the amount of critical information provided with papers reporting qPCR data is inadequate for the purpose of evaluating the validity of conclusions arising from those data, with many not reporting primer sequences, validation data or including wrong information.…”

Section: Introductionmentioning

confidence: 99%

qPCR primer design revisited

Bustin

Huggett

2017

Biomolecular Detection and Quantification

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165

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Primers are arguably the single most critical components of any PCR assay, as their properties control the exquisite specificity and sensitivity that make this method uniquely powerful. Consequently, poor design combined with failure to optimise reaction conditions is likely to result in reduced technical precision and false positive or negative detection of amplification targets. Despite the framework provided by the MIQE guidelines and the accessibility of wide-ranging support from peer-reviewed publications, books and online sources as well as commercial companies, the design of many published assays continues to be less than optimal: primers often lack intended specificity, can form dimers, compete with template secondary structures at the primer binding sites or hybridise only within a narrow temperature range. We present an overview of the main steps in the primer design workflow, with data that illustrate some of the unexpected variability that often occurs when theory is translated into practice. We also strongly urge researchers to report as much information about their assays as possible in their publications.

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Section: Introductionmentioning

confidence: 99%

qPCR primer design revisited

Bustin

Huggett

2017

Biomolecular Detection and Quantification

Self Cite

222

165

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“…We show that the analytical sensitivity of our assay is to the level of single cell RNA detection for relatively highly expressed RPL13a. Sample concentration and clean-up has been suggested to remove inhibitors and increase sensitivity (63,64). Unexpectedly, we found this step did not improve our analytical sensitivity, and was timeconsuming, expensive and reduced sample volume.…”

Section: Discussionmentioning

confidence: 80%

An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells

et al. 2020

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Reliable extraction and sensitive detection of RNA from human peripheral blood mononuclear cells (PBMCs) is critical for a broad spectrum of immunology research and clinical diagnostics. RNA analysis platforms are dependent upon high-quality and high-quantity RNA; however, sensitive detection of specific responses associated with high-quality RNA extractions from human samples with limited PBMCs can be challenging. Furthermore, the comparative sensitivity between RNA quantification and best-practice protein quantification is poorly defined. Therefore, we provide herein a critical evaluation of the wide variety of current generation of RNA-based kits for PBMCs, representative of several strategies designed to maximize sensitivity. We assess these kits with a reverse transcription quantitative PCR (RT-qPCR) assay optimized for both analytically and diagnostically sensitive cell-based RNA-based applications. Specifically, three RNA extraction kits, one post-extraction RNA purification/concentration kit, four SYBR master-mix kits, and four reverse transcription kits were tested. RNA extraction and RT-qPCR reaction efficiency were evaluated with commonly used reference and cytokine genes. Significant variation in RNA expression of reference genes was apparent, and absolute quantification based on cell number was established as an effective RT-qPCR normalization strategy. We defined an optimized RNA extraction and RT-qPCR protocol with an analytical sensitivity capable of single cell RNA detection. The diagnostic sensitivity of this assay was sufficient to show a CD8 + T cell peptide epitope hierarchy with as few as 1 × 10 4 cells. Finally, we compared our optimized RNA extraction and RT-qPCR protocol with current best-practice immune assays and demonstrated that our assay is a sensitive alternative to protein-based assays for peptide-specific responses, especially with limited PBMCs number. This protocol with high analytical and diagnostic sensitivity has broad applicability for both primary research and clinical practice.

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“…In a recent review about the use of RT-Q-PCR, Bustin and Nolan (Bustin & Nolan, 2017) stated that "the majority of published RT-Q-PCR data are likely to represent technical noise".…”

Section: Discussionmentioning

confidence: 99%

Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts

Cholet

Ijaz

Smith

2020

Preprint

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SummaryRT-Q-PCR, and RT-PCR amplicon sequencing, provide a convenient, target-specific, high-sensitivity approach for gene expression studies and are widely used in environmental microbiology. Yet, the effectiveness and reproducibility of the reverse transcription step has not been evaluated. Therefore, we tested a combination of four commercial reverse transcriptases with two priming techniques to faithfully transcribe 16S rRNA and amoA transcripts from marine sediments. Both enzyme and priming strategy greatly affected quantification of the exact same target with differences of up to 600-fold. Furthermore, the choice of RT system significantly changed the communities recovered. For 16S rRNA, both enzyme and priming had a significant effect with enzyme having a stronger impact than priming. Inversely, for amoA only the change in priming strategy resulted in significant differences between the same sample. Specifically, more OTUs and better coverage of amoA transcripts diversity were obtained with GS priming indicating this approach was better at recovering the diversity of amoA transcripts. Moreover, sequencing of RNA mock communities revealed that, even though transcript α diversities (i.e. OTU counts within a sample) can be biased by the RT, the comparison of β diversities (i.e. differences in OTU counts between samples) is reliable as those biases are reproducible between environments.Originality-Significance StatementIs the complementary DNA (cDNA) produced after Reverse Transcription (RT) a faithful representation of the starting RNA? This is a fundamental and important question for transcriptomic-based studies in environmental microbiology that aim to quantify and/or examine the diversity of transcripts via RT approaches. Yet little is known about the reliability and reproducibility of this step. Here, we evaluated the effect of the two main components of the RT reaction – the retro transcriptase enzyme and priming strategy (gene specific vs random priming), on the quantification and diversity of cDNA. We found that both have a significant impact. We further provide evidence to enable informed choices as to the enzyme and priming combinations to improve the performance of RT-PCR approaches. Taken together, this work will improve the reliability and reproducibility of transcript-based studies in environmental microbiology.

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Talking the talk, but not walking the walk: RT-qPCR as a paradigm for the lack of reproducibility in molecular research

Cited by 92 publications

References 161 publications

qPCR primer design revisited

qPCR primer design revisited

An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells

Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts

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