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Received on August 25, 2003
Accepted on December 23, 2003
Molecular Diagnostics and Genetics |
1 Department of Chemistry and Bioscience, Chalmers University of Technology, and TATAA Biocenter, Gothenburg, Sweden
2 Department of Medical Biochemistry, Gothenburg University, Gothenburg, Sweden
* To whom correspondence should be addressed. E-mail: mikael.kubista{at}tataa.com.
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 
-tubulin, glyceraldehyde-3-phosphate dehydrogenase, Glut2, CaV1D, and insulin II 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.
The following articles in journals at HighWire Press have cited this article:
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