Biopolym. Cell. 2016; 32(3):161-172.
http://dx.doi.org/10.7124/bc.00091A
Reviews
Practical approach to quantification of mRNA abundance using RT-qPCR, normalization of experimental data and MIQE
1Obolenskaya M. Yu., 1Kuklin A. V., 1Rodrigez R. R., 1Martsenyuk O. P., 1Korneyeva K. L., 1Docenko V. A., 1Draguschenko O. O.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680

Abstract

Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) has become the most common method for characterizing expression patterns of individual mRNAs due to a large dynamic range of linear quantification, high speed, sensitivity, resolution and cost-effectiveness. However, the complexity of the protocol, variability of reagents, an inconsistent quality of biological samples, and the absence of standardized methods of data quantification may produce inconsistent results. In an effort to to standardize ithe procedure and assure high reliability of data, the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines was defined and further extended by Prof. Bustin and colleagues (2004). These guidelines have been followed by the development of an XML-based real-time PCR data markup language (RDML) and a RDML data base for consistent reporting of RT-qPCR data created by the RDML consortium. Here we follow the RT-qPCR procedure step by step in compliance with MIQE requirements, local facilities and resources and our own experience in application of RT-qPCR methodology.
Keywords: RT-qPCR, normalization and standardization of data, MIQE
Full text: (PDF, in English)

References

[1] Rappolee DA, Mark D, Banda MJ, Werb Z. Wound macrophages express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping. Science. 1988;241(4866):708-12.
[2] Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611-22.
[3] Bustin SA, Beaulieu JF, Huggett J, Jaggi R, Kibenge FS, Olsvik PA, Penning LC, Toegel S. MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol. 2010;11:74.
[4] McNutt M. Journals unite for reproducibility. Science. 2014;346(6210):679.
[5] Leek JT, Peng RD. Statistics. What is the question? Science. 2015;347(6228):1314-5.
[6] Bustin SA. Molecular medicine, gene-expression profiling and molecular diagnostics: putting the cart before the horse. Biomark Med. 2008;2(3):201-7.
[7] Bustin SA, Benes V, Nolan T, Pfaffl MW. Quantitative real-time RT-PCR--a perspective. J Mol Endocrinol. 2005;34(3):597-601.
[8] Bustin S, Dhillon HS, Kirvell S, Greenwood C, Parker M, Shipley GL, Nolan T. Variability of the reverse transcription step: practical implications. Clin Chem. 2015;61(1):202-12.
[9] Joyce C. Quantitative RT-PCR. A review of current methodologies. Methods Mol Biol. 2002;193:83-92.
[10] Martin LA, Smith TJ, Obermoeller D, Bruner B, Kracklauer M, Dharmaraj S. RNA purification. In: Molecular Biology Problem Solver: A Laboratory Guide. Ed Alan S. Gerstein, 2001 by Wiley-Liss, Inc. Ch.8: 197-224.
[11] Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M. A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines. Methods. 2010;50(4):S1-5.
[12] Pfaffl MW. Quantification strategies in real-time PCR. In: A-Z of quantitative PCR (Editor: S.A. Bustin) International University Line (IUL) La Jolla, CA, USA. 2004; Chaper 3: 87–112.
[13] Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004;26(6):509-15.
[14] Lefever S, Hellemans J, Pattyn F, Przybylski DR, Taylor C, Geurts R, Untergasser A, Vandesompele J; RDML consortium. RDML: structured language and reporting guidelines for real-time quantitative PCR data. Nucleic Acids Res. 2009;37(7):2065-9.
[15] Lefever S, Vandesompele J, Speleman F, Pattyn F. RTPrimerDB: the portal for real-time PCR primers and probes. Nucleic Acids Res. 2009;37(Database issue):D942-5.
[16] Perepelyuk MM, Fedorchenko DB, Rybalko SL, Obolenskaya MYu. Interferon α expression in the rat liver after partial hepatectomy. Biopolym Cell 2006;22(4):276-282.
[17] Korneyeva KL, Rodrigues RR, Ralchenko SV, Martunovska OV, Frolova AO, Martsenyuk OP, Manzhula LV, Melnyk VT, Shkoropad OY, Obolenska MYu. Expression of genes, encoding the enzymes of cysteine metabolism in human placenta in the first and the third trimesters of gestation. Ukr Biochem J. 2016;8(1):88–98.
[18] Korneyeva KL, Rodrigues RR, Ralchenko SV, Vakulenko AV, Manzhula LV, Melnik VT, VereshchakOYu, ObolenskayaMYu. The expression of genes encoding the key enzymes of folate dependent metabolism in human placenta in first and third trimesters of uncomplicated pregnancy. Perinatologiya I pediatriya. 2014; 4(60):24–30.
[19] Kuklin AV, Tokovenko BT, Obolenska MYu. Evaluation criteria of rat hepatocytes transcriptome analysis under the influence of interferon alpha by DNA microarray. Biopolym Cell 2013; 29(6): 21–6.
[20] Kuklin AV, Poliezhaieva TA, Zhyryakova IO, Ogryzko VV, Obolenskaya MYu. Expression of ISGylation related genes in regenerating rat liver. Biopolym Cell. 2015; 31(5):351-61.
[21] Deng MY, Wang H, Ward GB, Beckham TR, McKenna TS. Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription-PCR. J Vet Diagn Invest. 2005;17(6):574-8.
[22] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. NY: Cold Spring Harbor Laboratory, 1989. 2nd ed.: vol. 3.
[23] Wilfinger WW, Mackey K, Chomczynski P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques. 1997;22(3):474-6, 478-81.
[24] Jones LJ, Yue ST, Cheung CY, Singer VL. RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterization. Anal Biochem. 1998;265(2):368-74.
[25] Imbeaud S, Graudens E, Boulanger V, Barlet X, Zaborski P, Eveno E, Mueller O, Schroeder A, Auffray C. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res. 2005;33(6):e56.
[26] Mueller O, Lightfoot S, and Schroeder A. RNA Integrity Number (RIN)—standardization of RNA quality control. 2004. Agilent Application Note, Publication Number-5989-1165EN, 1–8.
[27] Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol. 2006;7:3.
[28] Wiame I, Remy S, Swennen R, Sági L. Irreversible heat inactivation of DNase I without RNA degradation. Biotechniques. 2000;29(2):252-4, 256.
[29] Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc. 2006;1(2):581-5.
[30] Nolan T, Hands RE, Ogunkolade W, Bustin SA. SPUD: a quantitative PCR assay for the detection of inhibitors in nucleic acid preparations. Anal Biochem. 2006;351(2):308-10.
[31] Thornton B, Basu C. Real-time PCR (qPCR) primer design using free online software. Biochem Mol Biol Educ. 2011;39(2):145-54.
[32] Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003;31(13):3406-15.
[33] Yun JJ, Heisler LE, Hwang II, Wilkins O, Lau SK, Hyrcza M, Jayabalasingham B, Jin J, McLaurin J, Tsao MS, Der SD. Genomic DNA functions as a universal external standard in quantitative real-time PCR. Nucleic Acids Res. 2006;34(12):e85.
[34] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8.
[35] Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol. 2000;25(2):169-93.
[36] Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol. 2002;29(1):23-39.
[37] Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques. 2004;37(1):112-4, 116, 118-9.
[38] Huggett J, Dheda K, Bustin S, Zumla A. Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 2005;6(4):279-84.
[39] Kozera B, Rapacz M. Reference genes in real-time PCR. J Appl Genet. 2013;54(4):391-406.
[40] Wang GP, Xu CS. Reference gene selection for real-time RT-PCR in eight kinds of rat regenerating hepatic cells. Mol Biotechnol. 2010;46(1):49-57.
[41] Meller M, Vadachkoria S, Luthy DA, Williams MA. Evaluation of housekeeping genes in placental comparative expression studies. Placenta. 2005;26(8-9):601-7. ttp://
[42] Lion T. Current recommendations for positive controls in RT-PCR assays. Leukemia. 2001;15(7):1033-7.
[43] Barber RD, Harmer DW, Coleman RA, Clark BJ. GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues. Physiol Genomics. 2005;21(3):389-95.
[44] Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3(7):RESEARCH0034.
[45] Andersen CL, Jensen JL, Ørntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004;64(15):5245-50.
[46] Ruijter JM, Lefever S, Anckaert J, Hellemans J, Pfaffl MW, Benes V, Bustin SA, Vandesompele J, Untergasser A; RDML consortium. RDML-Ninja and RDMLdb for standardized exchange of qPCR data. BMC Bioinformatics. 2015;16:197.