Greetings!
I want to study ribosomal RNA expression and potential processing sites in bacteria. To such effect, I want to perform an RNA-Seq experiment in order to analyze potential rRNA transcripts isoforms and processing sites.
Conventional RNA-Seq experiments are not designed for the analysis of rRNA expression and generally include a RNA depletion step, which reduces rRNA signal. Notice rRNA in bacteria accounts for ~90% of total RNA.
In the context of studying rRNA processing, would the RNA-Seq approach be appropriate? If so, what rRNA treatment would you recommend to study rRNA expression levels and processing?"
Thank you in advance.
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Hi @Fernando,
the mir Vana miRNA Isolation Kit has demonstrated been useful for isolating rRNA. In order to study expression levels and processing probably Nanopore sequencing is the best alternative, since it allows accurate isoform quantification and eliminates PCR bias by using direct RNA sequencing.
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Hi there! I was thinking of skipping the rRNA depletion step, so nearly 90% of my RNA will be rRNA. Maybe the mir Vana miRNA Isolation Kit is more useful when the ribosomal portion of total RNA is smaller (e.g. humans)?
Thank you about the Nanopore suggestion, will definitely have a look at it
Hi @Fernando,
according to different publications , such as RNA Preservation Agents and Nucleic Acid Extraction Method Bias Perceived Bacterial Community Composition, this protocol should work fine with bacterial samples.
Regards
Sorry @gallardoalba, I see no why should this method work for the question I posed. Why not using samples without any rRNA isolation? I mean, if rRNA accounts for ~90% of total RNA then rRNA purification would not make such a big difference
Sorry @Fernando, I didn’t express it properly; the cited protocol can be used for extracting total RNA, not just rRNA. Any protocol for RNA extraction will be fine.
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Ok @gallardoalba thank you. My question quite goes in the sense of whether using a specific protocol for rRNA preservation or no protocol at all