ena-STUDY-UNIVERSITY OF GLASGOW-25-04-2019-14:38:24:288-264
Is the Reverse Transcription Step Reliable for Quantifying and Sequencing Bacterial Transcripts in Environmental Samples?
Measuring gene expression gives formidable insights into the mechanisms deployed by cells when facing immediate challenges. In microbiology, transcriptomics is essential to understanding which biochemical pathways are triggered when environmental conditions change. As RNA cannot directly be used as template, the transcriptomics workflow incorporates a step where the RNA is reverse transcribed (RT) into cDNA, which is then used for quantification (mostly Q-PCR) or sequencing. Even though indispensable, the variations and the impact of the RT step on end results has not been evaluated in environmental microbiology.In this second study we aimed at evaluating the effect of the reverse transcription reaction on the quantification (RT-Q-PCR) and sequencing (RT-PCR-sequencing) of Bacterial transcripts from environmental samples. A combination of four commonly used commercial reverse transcriptases (Superscript III, Superscript IV, Omniscript and Sensiscript) and two priming techniques (random hexamer and gene specific) were used. The effect of the different RT protocols was tested against endogenous transcripts and the results were validated using exogenous RNA spikes, for both RT-Q-PCR and RT-sequencing. Results obtained in this study show that, for the quantification and the sequencing of mRNA targets, Superscript IV and gene-specific priming was the best RT strategy. The pattern was not as clear for the 16SrRNA target, indicating a target-dependant behaviour of the RT step. Finally, the sequencing of RNA mock communities revealed that, even though transcript a diversities can be biased with some sequences being artificially over or under represented, the comparison of ß diversities is reliable as those biases are reproducible between environments.
Measuring gene expression gives formidable insights into the mechanisms deployed by cells when facing immediate challenges. In microbiology, transcriptomics is essential to understanding which biochemical pathways are triggered when environmental conditions change. As RNA cannot directly be used as template, the transcriptomics workflow incorporates a step where the RNA is reverse transcribed (RT) into cDNA, which is then used for quantification (mostly Q-PCR) or sequencing. Even though indispensable, the variations and the impact of the RT step on end results has not been evaluated in environmental microbiology.In this second study we aimed at evaluating the effect of the reverse transcription reaction on the quantification (RT-Q-PCR) and sequencing (RT-PCR-sequencing) of Bacterial transcripts from environmental samples. A combination of four commonly used commercial reverse transcriptases (Superscript III, Superscript IV, Omniscript and Sensiscript) and two priming techniques (random hexamer and gene specific) were used. The effect of the different RT protocols was tested against endogenous transcripts and the results were validated using exogenous RNA spikes, for both RT-Q-PCR and RT-sequencing. Results obtained in this study show that, for the quantification and the sequencing of mRNA targets, Superscript IV and gene-specific priming was the best RT strategy. The pattern was not as clear for the 16SrRNA target, indicating a target-dependant behaviour of the RT step. Finally, the sequencing of RNA mock communities revealed that, even though transcript a diversities can be biased with some sequences being artificially over or under represented, the comparison of ß diversities is reliable as those biases are reproducible between environments.