Artifacts and biases of the reverse transcription reaction in RNA-sequencing

Abstract

RNA sequencing has spurred a great number of research areas in recent years. Most protocols rely on converting RNA into a more stable complementary DNA copy during the reverse transcription reaction. The resulting cDNA pool is often wrongfully assumed to be quantitatively and molecularly similar to the original RN input. Sadly, biases and artifacts confound the resulting cDNA mixture. These issues are often overlooked or ignored in the literature by those that rely on the reverse transcription process. In this Review, we confront the reader with intra- and inter-sample biases, and artifacts caused by the reverse transcription reaction during RNA sequencing experiments. To fight the reader’s despair, we also provide solutions to most issues and inform on good RNA sequencing practices. We hope the reader can use this Review to his advantage, thereby contributing to scientifically sound RNA studies.