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Molecular Diagnostics and Genetics |
1 Department of Chemistry and Bioscience, Chalmers University of Technology, Gothenburg, Sweden. 2 TATAA Biocenter, 405 30 Gothenberg, Sweden. 3 Department of Medical Biochemistry, Gothenburg University, Gothenburg, Sweden.
aAddress correspondence to this author at: TATAA Biocenter, Medicinaregatan 7B, 405 30 Gothenburg, Sweden. Fax 46-31-7733948; 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 ß-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.
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