Ståhlberg Anders, Håkansson Joakim, Xian Xiaojie, Semb Henrik, Kubista Mikael
Department of Chemistry and Bioscience, Chalmers University of Technology, Gothenburg, Sweden.
Clin Chem. 2004 Mar;50(3):509-15. doi: 10.1373/clinchem.2003.026161. Epub 2004 Jan 15.
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.
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 II genes, using random hexamers, oligo(dT), and gene-specific reverse transcription primers.
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.
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.
在大多数基因表达测量中，mRNA首先被逆转录成cDNA。我们研究了逆转录反应及其对基因表达定量测量的影响。
我们使用基于SYBR绿I的定量实时PCR（QPCR），使用随机六聚体、寡聚（dT）和基因特异性逆转录引物，测量β-微管蛋白、甘油醛-3-磷酸脱氢酶、Glut2、CaV1D和胰岛素II基因的逆转录反应特性。
逆转录-QPCR（RT-QPCR）中的实验变异主要归因于逆转录步骤。逆转录效率取决于引物策略，且对于所研究的五个基因，这种依赖性有所不同。逆转录产量也取决于总RNA浓度。
只有当在所有实验中使用相同的引物策略和反应条件，且样品含有相同总量的RNA时，RT-QPCR基因表达测量结果才具有可比性。通过从逆转录反应开始（至少）一式两份运行样品，可提高实验准确性。
