The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

Huggett, Jim F.; Foy, Carole A.; Beneš, Vladimír; Emslie, Kerry R.; Garson, Jeremy A.; Haynes, Ross J.; Hellemans, Jan; Kubista, Mikael; Mueller, Reinhold; Nolan, Tania; Pfaffl, Michael W.; Shipley, Gregory L.; Vandesompele, Jo; Wittwer, Carl T.; Bustin, Stephen A.

doi:10.1373/clinchem.2013.206375

Cited by 762 publications

(681 citation statements)

References 43 publications

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“…Importantly, these parameters are based on common sense and current best practice and so are not set in stone and remain open for discussion; indeed, a slightly modifi ed version, labeled MIQE précis encompasses the key MIQE parameters essential for publication in Biomed Central (BMC) journals [ 11 ]. Most recently, MIQE-style guidelines for minimum information for publication of quantitative digital PCR experiments (dMIQE) have been published [ 12 ].…”

Section: Discussionmentioning

confidence: 99%

Minimum Information Necessary for Quantitative Real-Time PCR Experiments

Johnson

Nour

Nolan³

et al. 2014

Methods in Molecular Biology

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The MIQE (minimum information for the publication of quantitative real-time PCR) guidelines were published in 2009 with the twin aims of providing a blueprint for good real-time quantitative polymerase chain reaction (qPCR) assay design and encouraging the comprehensive reporting of qPCR protocols. It had become increasingly clear that variable pre-assay conditions, poor assay design, and incorrect data analysis were leading to the routine publication of data that were often inconsistent, inaccurate, and wrong. The problem was exacerbated by a lack of transparency of reporting, with the details of technical information inadequate for the purpose of assessing the validity of published qPCR data. This had, and continues to have serious implications for basic research, reducing the potential for translating fi ndings into valuable applications and potentially devastating consequences for clinical practice. Today, the rationale underlying the MIQE guidelines has become widely accepted, with more than 2,200 citations by March 2014 and editorials in Nature and related publications acknowledging the enormity of the problem. However, the problem we now face is rather serious: thousands of publications that report suspect data are populating and corrupting the peer-reviewed scientifi c literature. It will be some time before the many contradictions apparent in every area of the life sciences are corrected.

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Section: Discussionmentioning

confidence: 99%

Minimum Information Necessary for Quantitative Real-Time PCR Experiments

Johnson

Nour

Nolan³

et al. 2014

Methods in Molecular Biology

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“…Six microlitres of the eluted RNA samples were used for cDNA synthesis (Bioline Tetro cDNA Synthesis kit). For each genotype ddPCR (Droplet Digital PCR) was carried out on biological triplicates and quantified using the QX100 Droplet Digital PCR system (Bio-Rad) following the Digital MIQE guidelines 7,69 . The ddPCR mix consisted of: 10 ml 2 Â ddPCRt super mix for probes (Bio-Rad); 500 nM of primers for MYC and HFP, 250 nM of probe mix for MYC and HFP (Integrated DNA Technologies) and 1 ml of cDNA into a final volume of 20 ml.…”

Section: Methodsmentioning

confidence: 99%

Defective Hfp-dependent transcriptional repression of dMYC is fundamental to tissue overgrowth in Drosophila XPB models

et al. 2015

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Nucleotide excision DNA repair (NER) pathway mutations cause neurodegenerative and progeroid disorders (xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD)), which are inexplicably associated with (XP) or without (CS/TTD) cancer. Moreover, cancer progression occurs in certain patients, but not others, with similar C-terminal mutations in the XPB helicase subunit of transcription and NER factor TFIIH. Mechanisms driving overproliferation and, therefore, cancer associated with XPB mutations are currently unknown. Here using Drosophila models, we provide evidence that C-terminally truncated Hay/XPB alleles enhance overgrowth dependent on reduced abundance of RNA recognition motif protein Hfp/FIR, which transcriptionally represses the MYC oncogene homologue, dMYC. The data demonstrate that dMYC repression and dMYC-dependent overgrowth in the Hfp hypomorph is further impaired in the C-terminal Hay/XPB mutant background. Thus, we predict defective transcriptional repression of MYC by the Hfp orthologue, FIR, might provide one mechanism for cancer progression in XP/CS.

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“…16 The TaqMan KRAS_521 (Assay ID: AH6R5PI) assay was used for primers and probes. Reaction mixes (18 mL) were prepared, containing 9 mL twofold QuantStudio 3D Digital PCR Master Mix (Thermo Fisher Scientific), 0.9 mL 20-fold TaqMan KRAS_521 primer-probe mix, 1.8 mL diluted gDNA, and 6.3 mL nuclease-free water (Qiagen).…”

Section: Dpcrmentioning

confidence: 99%

Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer

Court

Ankeny

Sho

et al. 2016

The Journal of Molecular Diagnostics

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To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs. (J Mol Diagn 2016, 18: 688e696; http://dx

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The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

Cited by 762 publications

References 43 publications

Minimum Information Necessary for Quantitative Real-Time PCR Experiments

Minimum Information Necessary for Quantitative Real-Time PCR Experiments

Defective Hfp-dependent transcriptional repression of dMYC is fundamental to tissue overgrowth in Drosophila XPB models

Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer

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