Artifacts and biases of the reverse transcription reaction in RNA sequencing

RNA sequencing has spurred a significant number of research areas in recent years. Most protocols rely on synthesizing a more stable complementary DNA (cDNA) copy of the RNA molecule during the reverse transcription reaction. The resulting cDNA pool is often wrongfully assumed to be quantitatively and molecularly similar to the original RNA 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 intersample 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 their advantage, thereby contributing to scientifically sound RNA studies.

Automated summary

RNA sequencing has become influential in various research fields. However, the reverse transcription reaction in most protocols often leads to biases and artifacts in the resulting cDNA pool, which are overlooked in the literature. This review discusses these issues and provides solutions and good practices for RNA sequencing experiments, aiming to contribute to scientifically sound RNA studies.