User:Ajeffs/Notebook/Melanoma/2008/05/19

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The effect of PAX3 and MITF knock-down in melanoma cell lines

siRNA-mediated knockdown of PAX3 and MITF in NZM12 and NZM15

  • Reverse transfection with Dharmacon Smartpools (10 nM MITF; 20 nM PAX3; 20 nM non-targeting pool).
    1. Assess transcript knock-down at 24 hours post-txn by using qPCR.
    2. Assess protein levels 48 hours post-txn by using Western.
    3. n = 2 of 3 replicate experiments.
  • Measure EDNRB, RAB27A, FOXD3, and SNAI2 transcript levels by using qPCR at 24 and 48 hours post-txn.

Does miR-137 repeat expansion correlate with MITF levels in NZM cells?

  • Assess miR-137 locus stability at 1p22 with conventional PCR.
    1. purify and sequence a selection of the different sized amplicons.
    2. predict impact of repeat expansion on pre-miR folding.
    3. measure miR-137 VNTR status in non-melanoma/skin-derived cells?


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