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  • Question about what a particular Affymetrix microarray probeset is detecting

    Sorry not a sequencing question exactly, but I thought this might still be a good place to ask (is there a better place to ask microarray questions?).

    Most of the time if I get the target sequence for a probeset from the NetAffx website and then do a BLAT search the probeset aligns to the same strand as the transcript/gene it's detecting. For example, this one aligns to the "+" strand and detects the gene Cxcr2 which is also on the "+" strand:

    Code:
    >MOUSE430_2:1421734_AT
    gcatggcatcattaccagagactgtggtatttgaattgatgcagcccctcctctacatta
    cagggagaaagaggtcacgttcttcttagcagagccccccagagttttagaaccccctat
    atggctgtctgacccccttcatcattggtatgcctactgatagagttgatccatcctaac
    actgagaccccaaacactcttttctaagaagcacactttacaattacgtgagatctgcct
    ctacccatgcagaacagttagcagtaaaggaagaggtggaggagtagaaaatggcaagtg
    acaatgagaagtagaaaaagggaactcttcaccttaccagtagacgagtaccagagtccc
    ctcacacaggaaatagca
    This one aligns to the "-" strand and detects the gene Ntn1 which is also on the "-" strand:

    Code:
    >MOUSE430_2:1422987_AT
    gtgaacatcatctccgtgtacaagcagggcacaagtcgtattcgccgtggtgaccagagt
    ttgtggatccgctcacgagacatcgcctgcaagtgtcccaaaatcaagcccctcaagaag
    tacttgctgttgggtaatgccgaggactcacctgaccagagtggcatcgtggcagacaag
    agcagcctggtgatccagtggcgggacacatgggcacggcggctgcgcaagttccagcaa
    cgggagaagaagggcaagtgcaagaaggcctagcgcggaggtggcgcgggctccaggagg
    gcgggcagggcgctggcaaaggctggcagccttggacttggccgtcaggggttttttngg
    gagggtgggggcggggcgaagtcgaagtggggcggggccctcagcggctccgccccagac
    ccaccctcacacccctggctgcgctcttatgcgcatggcagaaagcaccctg
    This is an example of a probeset I'm having a problem with:

    Code:
    >MOUSE430_2:1446891_AT
    atcaactcctaccaatgttaccatgtaatttgataaaaatttgaatctttaactctgaaa
    gtgacctggaagttaaaaacacgatgggggaataaacacagaaagtgggtgtctgtgagt
    tcatcgtgctctacatagtgaaatcccaagacagcctaggctctggaaagagaccctatc
    tggaaaaaagattcccaagcaaataaatgaaactacttttaaactgttaacaggatacac
    ctcccagatgaaactattttgtagatttaagggagggaaaaaggctgtgatttatgagtt
    aaaccactgtttcatacatggttttggatattctaacctaaatgattacaaacaacctag
    aagagaagcaatctctttccttacctggctctggttaatagctgcatggttgccatggaa
    tggagactgcctttctttatctcggtggtgacgactgctgtgtttgcttctctgcagttg
    ctggcgtaattttgcaatctgaaa
    According to Affymetrix it detects the gene Fam117b, except that the probeset aligns to the "-" strand while the gene is on the "+" strand. So is it really detecting the Fam117b gene (and if so, how?), or is it actually detecting an antisense transcript that happens to be transcribed from within the Fam117b gene but from the opposite strand? Are microarrays strand-specific?

  • #2
    The Affymetrix gene expression microarrays like the one you are using are strand specific.

    The array will detect transcripts from whichever strand the probe sequences
    (target sequence) come from.

    If the gene is on the opposite strand to the probes, then it won't be detected by the array.

    Most of the probesets on the array will be annotated accurately, however,
    in cases where the annotation is based on a UniGene cluster, or where genes from different strands are close together, say at the 3' ends, then the annotation for the probeset can be problematic.

    Comment

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