Posted 03 June 2011 - 06:58 AM
Sure, thank you for your reply. In my protocol, the first half is the basic extraction of RNA from Trizol samples. After obtaining a pellet, it can be dissolved and quantified, stored, or one can continue on to the second half of the protocol which involves further purification through the use of several reagents and silica membrane spin columns. After I redissolved the pellet and quantified the RNA, I had high concentrations of RNA (~1000-1700ng/ul) as well as high 260/280 ratios but very low 260/230 ratios, indicating that further purification was necessary. I stored my samples in the -80C freezer until the spin columns arrived about a week later. I then thawed my samples on ice and proceeded through the rest of the protocol involving the addition of Buffer RLT and ethanol then transferring to the spin columns, etc. After completing the purification steps, I quantified the RNA again and expected high concentrations and high 260/280 and 260/230 ratios. However, the concentrations were much lower (<50ng/ul) and both ratios were very low. My reagents were no more than a couple days old and my samples had been carefully thawed on ice to prevent degradation. I have been researching the issue but have not found any answers. I simply do not understand why I am losing such large amounts of RNA. I have never had this issue when I perform the entire protocol without storage.