Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts.

Area of Science:

• Environmental microbiology
• Molecular biology
• Metagenomics

Background:

• Reverse-transcriptase quantitative PCR (RT-Q-PCR) and sequencing are vital for gene expression analysis in environmental microbiology.
• The critical reverse transcription (RT) step's impact on accuracy and reproducibility remains unevaluated.

Purpose of the Study:

• To assess the influence of different reverse transcriptase enzymes and priming techniques on the accurate transcription of 16S rRNA and amoA genes from marine sediments.
• To determine how RT system choices affect community composition and diversity measurements.

Main Methods:

• Tested four commercial reverse transcriptases with two priming strategies (random hexamers and gene-specific primers).
• Analyzed 16S rRNA and amoA transcripts from marine sediment samples.
• Sequenced RNA mock communities to evaluate biases in alpha and beta diversity.

Main Results:

• Both enzyme and priming significantly affected transcript quantification, with differences up to 600-fold.
• RT system choice altered recovered microbial communities.
• Gene-specific priming improved amoA transcript diversity recovery.
• While RT biases alpha diversity, beta diversity comparisons are reliable due to reproducible biases.

Conclusions:

• The reverse transcription step critically influences gene expression study outcomes in environmental microbiology.
• Careful selection of reverse transcriptase and priming strategy is essential for accurate quantification and community profiling.
• Despite potential biases, beta diversity analyses using RT-Q-PCR are robust for comparing microbial communities across different environments.