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How might I go about amplifying a gene transcript (mRNA) from animal tissue of which little is known about the genome? In some applications, I have used reverse transcriptase PCR to amplify all mRNA transcripts from cultured cells, but this required that I knew the sequence to PCR amplify.

How do I go about this without knowledge of the gene or transcipt?

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  • $\begingroup$ Do you know the function of the gene? What is the purpose of your experiment? $\endgroup$
    – Gergana Vandova
    Commented Mar 22, 2012 at 2:16
  • $\begingroup$ I know it's an endocrine hormone, and I can draw parallels from human endocrinology, but beyond that I don't know much about it (and there doesn't seem to be much in the literature either). The purpose really is just to see how conserved the mammalian orthologs are compared to lizards. $\endgroup$
    – user560
    Commented Mar 22, 2012 at 2:26
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    $\begingroup$ Having the hormone sequence does help a bit. While not entirely unbiased, you could try to enrich your sequence by amplifying from a conserved region. $\endgroup$
    – bobthejoe
    Commented Mar 22, 2012 at 3:51
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    $\begingroup$ Ya, bobthejoe is right: just search in google 'homology-based cloning' $\endgroup$
    – Gianpaolo R
    Commented Mar 22, 2012 at 8:14
  • $\begingroup$ @bobthejoe - thanks, I will look up homology-based cloning. I suspect there's a fair amount (50%) of conservation between humans and lizards so I'll see if this pathway can yield a meaningful result. $\endgroup$
    – user560
    Commented Mar 22, 2012 at 12:10

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Perhaps you can draw inspiration from classic paper on lambda cloning: Maniatis T, Hardison RC, Lacy E, Lauer J, O’Connell C, Quon D, Sim GK, Efstratiadis A. 1978. The isolation of structural genes from libraries of eucaryotic DNA. Cell 15: 687–701.

Try selecting tissues from the animal which you think is "enriched" (i.e. highly expressed) for the specific gene you're looking for. This might take a little bit of guessing. For example, if gene A is very highly expressed in tissue B, then you can "assume" most of the mRNA transcripts will be for that of gene A. Do a RNA purification, RT-PCR, and clone the cDNA into lambda vectors and pick plaques. With a little bit of luck, you'll be able to identify the gene by cloning, hybridisation, and subsequent chromosome "walking" to determine it's structural origin.

This was done back in the day to identify novel genes way before genomics, sequencing, or even PCR!

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  • $\begingroup$ That's pretty cool. Thanks for the reference and explanation jp89. $\endgroup$
    – user560
    Commented Mar 22, 2012 at 12:09

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