Improving the Standardization of MRNA Measurement by RT-qPCR

Overview

Journal Biomol Detect Quantif

Date 2018 Jun 21

PMID 29922589

Citations 9

Authors

Rebecca Sanders

Stephen Bustin

Jim Huggett

Deborah Mason

Affiliations

Soon will be listed here.

Abstract

Human health and safety depend on reliable measurements in medical diagnosis and on tests that support the selection and evaluation of therapeutic intervention and newly discovered molecular biomarkers must pass a rigorous evaluation process if they are to be of benefit to patients. Measurement standardization helps to maximize data quality and confidence and ultimately improves the reproducibility of published research. Failure to consider how a given experiment may be standardized can be costly, both financially as well as in time and failure to perform and report pre-clinical research in an appropriately rigorous manner will hinder the development of diagnostic methods. Hence standardization is a crucial step in maintaining the integrity of scientific studies and is a key feature of robust investigation.

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References

Bohlenius H, Eriksson S, Parcy F, Nilsson O . Retraction. Science. 2007; 316(5823):367. DOI: 10.1126/science.316.5823.367b. View

Lefever S, Hellemans J, Pattyn F, Przybylski D, Taylor C, Geurts R . RDML: structured language and reporting guidelines for real-time quantitative PCR data. Nucleic Acids Res. 2009; 37(7):2065-9. PMC: 2673419. DOI: 10.1093/nar/gkp056. View

Maess M, Sendelbach S, Lorkowski S . Selection of reliable reference genes during THP-1 monocyte differentiation into macrophages. BMC Mol Biol. 2010; 11:90. PMC: 3002353. DOI: 10.1186/1471-2199-11-90. View

Aursnes I, Rishovd A, Karlsen H, Gjoen T . Validation of reference genes for quantitative RT-qPCR studies of gene expression in Atlantic cod (Gadus morhua l.) during temperature stress. BMC Res Notes. 2011; 4:104. PMC: 3080820. DOI: 10.1186/1756-0500-4-104. View

Dijkstra J, van Kempen L, Nagtegaal I, Bustin S . Critical appraisal of quantitative PCR results in colorectal cancer research: can we rely on published qPCR results?. Mol Oncol. 2014; 8(4):813-8. PMC: 5528534. DOI: 10.1016/j.molonc.2013.12.016. View

Arun A, Baumle V, Amelot G, Nieberding C . Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana. PLoS One. 2015; 10(3):e0120401. PMC: 4368739. DOI: 10.1371/journal.pone.0120401. View

Devonshire A, Sanders R, Whale A, Nixon G, Cowen S, Ellison S . An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1. Biomol Detect Quantif. 2016; 8:15-28. PMC: 4906133. DOI: 10.1016/j.bdq.2016.05.003. View

Rutledge R . Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications. Nucleic Acids Res. 2004; 32(22):e178. PMC: 545475. DOI: 10.1093/nar/gnh177. View

Rutledge R, Stewart D . Assessing the performance capabilities of LRE-based assays for absolute quantitative real-time PCR. PLoS One. 2010; 5(3):e9731. PMC: 2840021. DOI: 10.1371/journal.pone.0009731. View

10.

Perez-Novo C, Claeys C, Speleman F, van Cauwenberge P, Bachert C, Vandesompele J . Impact of RNA quality on reference gene expression stability. Biotechniques. 2005; 39(1):52, 54, 56. DOI: 10.2144/05391BM05. View

11.

Nielsen K, Mogensen H, Hedman J, Niederstatter H, Parson W, Morling N . Comparison of five DNA quantification methods. Forensic Sci Int Genet. 2008; 2(3):226-30. DOI: 10.1016/j.fsigen.2008.02.008. View

12.

Brazma A, Hingamp P, Quackenbush J, Sherlock G, Spellman P, Stoeckert C . Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet. 2001; 29(4):365-71. DOI: 10.1038/ng1201-365. View

13.

Belder N, Coskun O, Doganay Erdogan B, Ilk O, Savas B, Ensari A . From RNA isolation to microarray analysis: Comparison of methods in FFPE tissues. Pathol Res Pract. 2016; 212(8):678-85. DOI: 10.1016/j.prp.2015.11.008. View

14.

Valasek M, Repa J . The power of real-time PCR. Adv Physiol Educ. 2005; 29(3):151-9. DOI: 10.1152/advan.00019.2005. View

15.

Chapuis M, Tohidi-Esfahani D, Dodgson T, Blondin L, Ponton F, Cullen D . Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust. BMC Mol Biol. 2011; 12:7. PMC: 3048552. DOI: 10.1186/1471-2199-12-7. View

16.

Nolan T, Hands R, Bustin S . Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2007; 1(3):1559-82. DOI: 10.1038/nprot.2006.236. View

17.

Huggett J, Novak T, Garson J, Green C, Morris-Jones S, Miller R . Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon. BMC Res Notes. 2008; 1:70. PMC: 2564953. DOI: 10.1186/1756-0500-1-70. View

18.

Metcalf D, Weese J . Evaluation of commercial kits for extraction of DNA and RNA from Clostridium difficile. Anaerobe. 2012; 18(6):608-13. DOI: 10.1016/j.anaerobe.2012.10.003. View

19.

Takahashi K, Ikeda N, Nonoguchi N, Kajimoto Y, Miyatake S, Hagiya Y . Enhanced expression of coproporphyrinogen oxidase in malignant brain tumors: CPOX expression and 5-ALA-induced fluorescence. Neuro Oncol. 2011; 13(11):1234-43. PMC: 3199158. DOI: 10.1093/neuonc/nor116. View

20.

Spiess A, Feig C, Ritz C . Highly accurate sigmoidal fitting of real-time PCR data by introducing a parameter for asymmetry. BMC Bioinformatics. 2008; 9:221. PMC: 2386824. DOI: 10.1186/1471-2105-9-221. View