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  • Best mRNA isolation method for grasses

    Hi all,

    I am new to the sequencing world and want to sequence a grass cDNA library using the 454 Junior. Can anyone suggest a good method for mRNA isolation? A collegue uses RNAzol for qPCR...any experience with this for sequencing?

    Also, what about normalization...how necessary is this if we expect our genes of interest to be highly expressed?

  • #2
    Regarding mRNA isolation, I would recommend RNeasy Plant Mini Kit (QIAGEN) and then PolyATtract mRNA Isolation System (Promega) for large quantity mRNA purification or Dynabeads mRNA DIRECT Micro Kit (Dynal/Invitrogen) for small scale purification. I am working on barley.

    Next week I will start with cDNA sequencing on Junior. I am doing normalization by re-association first and then polyA trimming.

    bests,
    mk

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    • #3
      Thanks, audioPL. I haven't had great yield with the RNA easy kit. I am trying Trizol, which we normally use for total RNA, and I am indeed trying the PolyAtract kit. We've decided not to normalize for this run, but may in the future so thanks for your tip there as well.

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      • #4
        We have used RNeasy plant mini kit for microarray expermients and it worked well. I'm working on maize.

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        • #5
          I have always stuck with Trizol, you usually get fairly good yield with that and one simply looses so much with kits. Unless you are processing a large number of samples, I don't see significant advantage in using a kit. I will on occasion use RNAeasy (just the regular kit) for cleanup after DNAse treatment.

          Actually, my bigger concern would be whether or not the 454 Junior is the right tool for the job. The throughput is rather low, particularly for a plant transcriptome...which can be much larger than animal transcriptomes due to the commonality of whole genome duplication.

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          • #6
            I am with chadn737 on that.
            You will not be sequencing very deep into the complexity of the pool on non-normalized 454 cDNA. However, maybe that is inherent in the design. You are likely not shooting for comparative expression of mid expressed genes.

            We have used the 454 low coverage sequencing to greatly improve the transcriptome assembly of our illumina data (ie mixed platform approach).

            Best of luck.
            j

            Jarret Glasscock
            Cofactor Genomics

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