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  • Help for experimental design

    We proposed to detect the transcriptome/mirnaome of inner cell mass of porcine.The bottleneck for this study is that we could get very few cells ( about 10 from each blastocyst,~10 blastocyst for each fertilized porcine.We prepared 5 pigs for this experiment. So, we could get ~ 500 cells. ) Is so many cells enough for transcriptome/mirnaome analysis by Illumina Hiseq 2000? What else is worthy of note?

  • #2
    Here are a couple links to resources for amplifying small amounts of RNA. I have used the first two, and they work well.

    Nugen Ovation RNA-Seq:
    http://www.nugeninc.com/nugen/index....na-seq-system/

    Modified Illumina RNA-Seq protocol (look in the supplemental data):
    http://www.plosbiology.org/article/i...l.pbio.1000590

    Linear amplification of RNA:
    http://www.jove.com/details.stp?id=2634

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    • #3
      DSN protocol really works. We used non detectable amount of total RNA as an input for library prep and got ~30M passing reads from single GAIIx lane. Unique read was ~5M, so higher redundancy as you expected from extremely low input material, but this was great!

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