The Ultimate qPCR Experiment: Producing Publication Quality, Reproducible Data the First Time

被引：552

作者：

Taylor, Sean C. ^{[1
]}

Nadeau, Katia ^{[1
]}

Abbasi, Meysam ^{[1
]}

lachance, ClauDe ^{[1
]}

Nguyen, Marie ^{[2
]}

Fenrich, Joshua ^{[2
]}

机构：

[1] Biorad Labs Canada Inc, 1329 Meyerside Dr, Mississauga, ON L5T1C9, Canada

[2] Biorad Labs, 255 Linus Pauling Dr, Hercules, CA 94547 USA

来源：

TRENDS IN BIOTECHNOLOGY | 2019年 / 37卷 / 07期

关键词：

DROPLET DIGITAL PCR; MINIMUM INFORMATION; QUANTITATIVE PCR; RT-QPCR; RNA; DNA; OPTIMIZATION; PLANT; TRANSCRIPTION; VARIABILITY;

D O I：

10.1016/j.tibtech.2018.12.002

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the methodology is perceived to be relatively simple, there are a number of steps and reagents that require optimization and validation to ensure reproducible data that accurately reflect the biological question(s) being posed. This review article describes and illustrates the critical pitfalls and sources of error in qPCR experiments, along with a rigorous, stepwise process to minimize variability, time, and cost in generating reproducible, publication quality data every time. Finally, an approach to make an informed choice between qPCR and digital PCR technologies is described.

引用

页码：761 / 774

页数：14

共 63 条

[1]

Albert KJ., 2007, JALA: J Assoc Lab Autom, V12, P172, DOI [10.1016/j.jala.2006.10.005, DOI 10.1016/J.JALA.2006.10.005]

[2] Information bias in health research: definition, pitfalls, and adjustment methods [J].

Althubaiti, Alaa .

JOURNAL OF MULTIDISCIPLINARY HEALTHCARE, 2016, 9 :211-217

[3] Properties of targeted preamplification in DNA and cDNA quantification [J].

Andersson, Daniel ;

Akrap, Nina ;

Svec, David ;

Godfrey, Tony E. ;

Kubista, Mikael ;

Landberg, Goran ;

Stahlberg, Anders .

EXPERT REVIEW OF MOLECULAR DIAGNOSTICS, 2015, 15 (08) :1085-1100

[4] Bleach gel: A simple agarose gel for analyzing RNA quality [J].

Aranda, Patrick S. ;

LaJoie, Dollie M. ;

Jorcyk, Cheryl L. .

ELECTROPHORESIS, 2012, 33 (02) :366-369

[5] Control of Transcript Variability in Single Mammalian Cells [J].

Battich, Nico ;

Stoeger, Thomas ;

Pelkmans, Lucas .

CELL, 2015, 163 (07) :1596-1610

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

Bustin, Stephen ;

Nolan, Tania .

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, 2017, 47 (10) :756-774

[7] Variability of the Reverse Transcription Step: Practical Implications [J].

Bustin, Stephen ;

Dhillon, Harvinder S. ;

Kirvell, Sara ;

Greenwood, Christina ;

Parker, Michael ;

Shipley, Gregory L. ;

Nolan, Tania .

CLINICAL CHEMISTRY, 2015, 61 (01) :202-212

[8]

Bustin Stephen A, 2017, Biomol Detect Quantif, V11, P1, DOI 10.1016/j.bdq.2017.01.002

[9] The need for transparency and good practices in the qPCR literature [J].

Bustin, Stephen A. ;

Benes, Vladimir ;

Garson, Jeremy ;

Hellemans, Jan ;

Huggett, Jim ;

Kubista, Mikael ;

Mueller, Reinhold ;

Nolan, Tania ;

Pfaffl, Michael W. ;

Shipley, Gregory ;

Wittwer, Carl T. ;

Schjerling, Peter ;

Day, Philip J. ;

Abreu, Monica ;

Aguado, Begona ;

Beaulieu, Jean-Francois ;

Beckers, Anneleen ;

Bogaert, Sara ;

Browne, John A. ;

Carrasco-Ramiro, Fernando ;

Ceelen, Liesbeth ;

Ciborowski, Kate ;

Cornillie, Pieter ;

Coulon, Stephanie ;

Cuypers, Ann ;

De Brouwer, Sara ;

De Ceuninck, Leentje ;

De Craene, Jurgen ;

De Naeyer, Helene ;

De Spiegelaere, Ward ;

Deckers, Kato ;

Dheedene, Annelies ;

Durinck, Kaat ;

Ferreira-Teixeira, Margarida ;

Fieuw, Annelies ;

Gallup, Jack M. ;

Gonzalo-Flores, Sandra ;

Goossens, Karen ;

Heindryckx, Femke ;

Herring, Elizabeth ;

Hoenicka, Hans ;

Icardi, Laura ;

Jaggi, Rolf ;

Javad, Farzad ;

Karampelias, Michael ;

Kibenge, Frederick ;

Kibenge, Molly ;

Kumps, Candy ;

Lambertz, Irina ;

Lammens, Tim .

NATURE METHODS, 2013, 10 (11) :1063-1067

[10] The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments [J].

Bustin, Stephen A. ;

Benes, Vladimir ;

Garson, Jeremy A. ;

Hellemans, Jan ;

Huggett, Jim ;

Kubista, Mikael ;

Mueller, Reinhold ;

Nolan, Tania ;

Pfaffl, Michael W. ;

Shipley, Gregory L. ;

Vandesompele, Jo ;

Wittwer, Carl T. .

CLINICAL CHEMISTRY, 2009, 55 (04) :611-622

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