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The importance and role of reverse transcriptases in gene expression analysis
Žucha, Daniel ; Valihrach, Lukáš (advisor) ; Španielová, Hana (referee)
The continuously advancing field of gene expression analysis enables the evaluation of even the slightest changes that occur in the cell transcriptome. In order to ensure accuracy of the observed biological variances, it is fundamentally important to be aware of the possible biases introduced during sample processing. In gene expression research, the methods of reverse transcription−quantitative PCR (RT−qPCR) and RNA- Sequencing (RNA-Seq) are often the primary choice, mostly because of their high precision and reproducibility. Since these both methods require DNA template, they are coupled with the same initial step - reverse transcription (RT), a reaction producing DNA complementary to its RNA template. It is well known that RT introduces bias. As a result, it is therefore of importance to thoroughly evaluate the effects of these biases. One such annotated source of artifacts is the reverse transcriptase (RTase) itself. However, it has been shown that the enzyme does not account for most of the variance alone. Surprisingly, choice of primers or RNA template may influence the reaction outcome even more than the bias introduced from the enzyme. This is especially the case with recent advances in protein engineering. Production of highly efficient RTases may pronounce the variation originating from...
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