- 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..