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  • pmiguel
    replied
    Hence the "well actually..." prefix -- a signal that speaker is about to bring up some pedantic correction. However I did think it worth mentioning because I've had cases where we got eukaryotic mRNA samples that were contaminated with bacterial RNA. Then some of that RNA ends up getting sequenced.

    In one case the investigator that did the RNA prep even admitted that the samples (leaves) were a bit slimy prior to isolation. But they objected that the bacterial RNA should not have ended up in the sequence because it was poly A+ selected and bacterial RNA was not polyadenylated. At the time I thought this was true and was puzzled as well. But subsequently at a bacterial genomics meeting here at Purdue I learned that bacteria did polyadenylate their RNA.

    So, worth keeping in mind...

    --
    Phillip

    Leave a comment:


  • protist
    replied
    Originally posted by pmiguel View Post
    Well actually... some bacterial RNAs are poly-adenylated. A google search for "bacterial RNA polyadenylation" will give you the basics.

    But your main point -- polyA+ purification is not an appropriate method for isolating mRNA from bacteria is correct.

    --
    Phillip
    Yes a google search will give you the basics and further reading will reveal that bacterial polyA transcripts are more often than not destined for degradation. The marker for bacterial mRNA is the presence of a triphosphate.

    Your second point is also true as a polyA selection of bacterial RNA will not capture its transcriptome and is more likely reflective of the its RNA degradome.

    Leave a comment:


  • pmiguel
    replied
    Originally posted by protist View Post
    For bacterial RNAseq we always assay the whole transcriptome as our only option is to rRNA deplete as bacterial mRNAs do not have polyA tails.
    Well actually... some bacterial RNAs are poly-adenylated. A google search for "bacterial RNA polyadenylation" will give you the basics.

    But your main point -- polyA+ purification is not an appropriate method for isolating mRNA from bacteria is correct.

    --
    Phillip

    Leave a comment:


  • protist
    replied
    Originally posted by mathew View Post
    I am little confused in one of the commercial tool Avadis there are to RNA-seq work flows one named mRNA-seq and second is whole genome RNA-seq. Tried to search online or tools resources but could not find a good answer.
    Any pointers will be greatly appreciated.

    Thanks
    Whole genome RNAseq is the term often used for libraries where there has been ribosomal RNA depletion but no polyA selection - in this case RNAs that are not polyadenlated are retained in the library thus it can be categorized as whole transcriptome view - mRNA(polyA component) and various other non polyA RNA species such as small RNAs or non-coding RNAs are assayed.

    mRNAseq term is often used for libraries which have been constructed from polyA selected material only ie the mRNA component of eukaryotes.

    For bacterial RNAseq we always assay the whole transcriptome as our only option is to rRNA deplete as bacterial mRNAs do not have polyA tails.

    Leave a comment:


  • mathew
    started a topic Whole genome RNA-seq and mRNAseq

    Whole genome RNA-seq and mRNAseq

    I am little confused in one of the commercial tool Avadis there are to RNA-seq work flows one named mRNA-seq and second is whole genome RNA-seq. Tried to search online or tools resources but could not find a good answer.
    Any pointers will be greatly appreciated.

    Thanks

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