Properties of the reverse transcription reaction in mRNA quantification

被引:277
作者
Ståhlberg, A
Håkansson, J
Xian, XJ
Semb, H
Kubista, M
机构
[1] TATAA Bioctr, S-40530 Gothenburg, Sweden
[2] Chalmers Univ Technol, Dept Chem & Biosci, S-41296 Gothenburg, Sweden
[3] Gothenburg Univ, Dept Med Biochem, Gothenburg, Sweden
关键词
D O I
10.1373/clinchem.2003.026161
中图分类号
R446 [实验室诊断]; R-33 [实验医学、医学实验];
学科分类号
1001 ;
摘要
Background: In most measurements of gene expression, mRNA is first reverse-transcribed into cDNA. We studied the reverse transcription reaction and its consequences for quantitative measurements of gene expression. Methods: We used SYBR green I-based quantitative real-time PCR (QPCR) to measure the properties of reverse transcription reaction for the beta-tubulin, glyceraldehyde-3-phosphate dehydrogenase, Glut2, CaV1D, and insulin 11 genes, using random hexamers, oligo(dT), and gene-specific reverse transcription primers. Results: Experimental variation in reverse transcription-QPCR (RT-QPCR) was mainly attributable to the reverse transcription step. Reverse transcription efficiency depended on priming strategy, and the dependence was different for the five genes studied. Reverse transcription yields also depended on total RNA concentration. Conclusions: RT-QPCR gene expression measurements are comparable only when the same priming strategy and reaction conditions are used in all experiments and the samples contain the same total amount of RNA. Experimental accuracy is improved by running samples in (at least) duplicate starting with the reverse transcription reaction. (C) 2004 American Association for Clinical Chemistry.
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页码:509 / 515
页数:7
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