RNA purification from pancreas - troubles with the pancreas (Jun/19/2007 )
Hello, all.
I'm looking for some advice regarding the extraction of total RNA from pancreas. I use RNAlater for stabilizing the tissue ( apply it directly on the pancreas) and afterwards homogenize it and extract it in peqGOLD TRIFAST reagent. After the steps of chloroform, isopropranol and 75%EtOH, I get some readings of RNA conc.(800-2000µg/ml) and most of the times low 260/280 ratio (1.7 or less). I tried re-purifying it with the Trifast and/or additional chloroform. After the RT-PCR I don't get any products in my experiments. In the same time, RNA from testis processed in the same way (my positive control) works just fine.
My idea is that my pancreatic RNA is being degraded at some point...
So I`m asking for some ideas, solutions, hints from you. Anybody having some experience with pancreatic RNA purification?
Thank you very much in advance
I'm looking for some advice regarding the extraction of total RNA from pancreas. I use RNAlater for stabilizing the tissue ( apply it directly on the pancreas) and afterwards homogenize it and extract it in peqGOLD TRIFAST reagent. After the steps of chloroform, isopropranol and 75%EtOH, I get some readings of RNA conc.(800-2000µg/ml) and most of the times low 260/280 ratio (1.7 or less). I tried re-purifying it with the Trifast and/or additional chloroform. After the RT-PCR I don't get any products in my experiments. In the same time, RNA from testis processed in the same way (my positive control) works just fine.
My idea is that my pancreatic RNA is being degraded at some point...
So I`m asking for some ideas, solutions, hints from you. Anybody having some experience with pancreatic RNA purification?
Thank you very much in advance
Hi McJoseph, I don't have direct experience with RNA isolation from the pancreas but others have suggested that there are a lot of RNases. However I wouldn't jump to the conclusion that you are getting degradation just because the 260/280 ratio is low until you check out the integrity via northern (28S and 18S). If you see minimal degradation (band smearing) I would try acid phenol/ chloroform extraction followed by DNase digest or use the Ambion RNA kits. If it's degradation, then I would skip the RNAlater step and get the pancreas into Trizol and homogenize immediately after isolation. If for whatever reason you can't do this, then snap freeze in liquid nitrogen.