I am trying to isolate RNA from Breast tumor and adjacent normal tissues (stored in RNAlater at -20 C) using Trizol method. After adding isopropanol , I am getting a transparent droplet like pellet that does not stick to the bottom. And there are no visible bands on gel either.
before starts remove RNAlater from the tissue surface by washing in a nuclease free water then for 100mg tissue add 600-750 mL trizol and crush with micro pestle, spin to remove tissue debris, separate the sup in a fresh vial wait for 2 minutes, add 200uL of chloroform, mix vigorously until pink color, wait for 3-4 minutes at RT and rest are the same, but don,t over dry the pellet after 70% ethanol wash and dissolve in a RNAstorage solution.
Hi ... I've used the same tissue and method u are trying ..... but i think u should cut the tumor from solid part ... u may cut part from the fatty tissues
Good luck
Hello Bhoomika Sharma,
I need your help. I am facing similar problem like you. In my case RNA concentration around 500-600 ng/ul, and purity is also relatively good. After making cDNA, when I performed the real time PCR for gene expression, I couldn't find a good dissociation curve even for house keeping gene. How did you solve your problem? Could you please share with me. Thank you very much.
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