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  • #16
    short genes

    hello cole and epi,

    i have made some comparisons of FPKM values (calculated from count data, not with cufflinks) with corresponding microarray data regarding gene length, and found some interesting details.

    it is true that FPKM values from genes with length below 500 bp correlate much less with expression values derived from microarrays, but the differences between tissues (i.e. normal vs. cancer) from small genes correlated even better (NGS vs microarray) than the differences from larger genes (interestingly, the correlations go continuos down. see figure).

    and in most study designs, the differences from two conditions are important, not the absolute expression values. therefore, i would not exclude small genes from statistical analysis!

    in green and blue are the correlations of lg2_FPKM values from each 12 normal and 12 cancer tissues with corresponding lg2_microarray_expression values from 26 normal and 26 cancer tissues. in red the correlations of the differences (lg2_FPKM_NORMAL - lg2_FPKM_CANCER vs. lg2_microarray_NORMAL - lg2_microarray_CANCER) are shown. on the x-axis genes are grouped according gene length (and the number of genes in each bin are shown), e.g. 190 genes are below 500 bp length.
    Attached Files

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    • #17
      @dietmar

      What method did you use to calculate expression on the microarrays and what kind of microarrays were they?

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      • #18
        @arvid

        publicly available: GSE25070

        http://www.ncbi.nlm.nih.gov/projects...i?acc=GSE25070

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        • #19
          Originally posted by dietmar13 View Post
          Thanks! Is the corresponding RNA-seq data available too by any chance?

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          • #20
            steven, you are lucky,

            both data are not from us, i am only playing around...

            12 normal vs 12 colon cancer, paired:
            sra:
            SRP007584

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            • #21
              Small RNA

              So that means Cufflinks cannot be used for micro RNA or small RNAs. Any one differ on this please.

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              • #22
                I think first of all, it means that microRNA and smallRNA cannot be studied by in canonical RNA-Seq experiment, aimed at studying "longer" RNA (mRNA, lincRNA).
                For smallRNA, you do not need to assemble spliced transcripts, and therefore, you could use another type of analysis: something along the lines of:
                1) remove the adapter
                2) map with Bowtie/BWA
                3) count the reads mapping to miRBase hairpins + your smallRNA genes of interest

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