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Can RNase inactivation takes place at 65 Degree for 48 h, incubation.

RNase inactivation hybridization

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#1 abpatel2288

abpatel2288

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Posted 28 November 2011 - 04:43 AM

I am trying to do cDNA-RNA hybridization (subtractive hybridization) at 65 degree for 48 hrs,
  • i am isolation RNA from two different tissue say liver and muscle.
  • than i am synthesizing 1st strand cDNA of liver RNA with oigo-dT (labeled), followed by addition of oligo A and finally purifying cDNA with column.
  • Than using (liver) cDNA and (muscle) RNA in 1:1, 1:3 and 1:3 ratio of cDNA and RNA i am proceeding for hybridization(in 1X SSPE and Denhardt hybridization buffer at 65 degree 48 hours.
  • On completion of above step i am adding streptavidin beads, so that labeled cDNA will bind to it, along with RNA which have hybridized to it.
  • than i am trying to concentrate UN-hybridized RNA from supernatant, but i am not getting RNA...


IT seems that RNA is degraded at 65 degree either due to RNase activity or due temperature...


Please suggest modifications to carry out such hybridization process..





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