I am trying to isolate a mRNA (1400 bases) from Dog placenta with trizol reagent. Every time we are getting degraded RNA. We have stored the 100mg sample in 300ul trizol reagent and stored at -80. what are the possible reasons for the degradation of the rna in the sample ? How to overcome this problem ?
I have uploaded gel picture with Thermo 1kb dna ladder.
Dear Perumal,
300 µl Trizol is not a sufficient amount for 100 mg tissue. You must use at least 1 ml Trizol as it is stated in Thermo's protocol. That's one possibility for the degradation. Additionally some of your reagents might be contaminated with RNases. You should use a fresh batch of everything expecially your elution buffer (Water) should be made with DEPC treated water. In addition make sure you follow guidelines for RNase-free working. You may consider a clean-up step with silica membranes. Degradation can also occur in the gel so make sure you add some RNase-Inhibitor to your loading sample.
Best regards.
Thank you for your kind reply Dr.Soner Öner-Sieben. Actually am usig 300 µl Trizol for storage purpose at -80 C But for RNA isolation we were using 1ml of trizol. we ll follow above said protocol.
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