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[abpatel2288]

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..