My RNA extraction failed (From a newbie ) - (Jun/13/2012 )
I am quite newbie in molecular biology, and i'm just learning how to extract RNA from my tissue samples (mice dental pulp) in order to , in future, can do PCR analysis.
i followed all the steps of the TRIZOL protocol for RNA extraction but when i did the reading of the concentration using the spectrophotometer those values were really low! 69 ug/ml or so...quite different to those obtained by my supervisor, up to 2000 ug/ml
After I took the dental pulp of mice (more than one week ago) I storaged them in trizol *1ml* at -80C , then I defrost at room temperature and homogenized by pippeting and normal vortex...I recognize I have probably taken some of the interphase together with the aquous phase after adding chloroform ...but according to one senior it should'nt be the reason for such low values. I could observe the precipitation even after 70% ethanol washing...Is this problem related to the sample?
Any suggestion would be appreciated
Which TRIZOL extraction are you using? Is this a TRIZOL LS method from Invitrogen? In my experience, TRIZOL gives me high reading yield, but the RNA is not so clean. However, there is a chloroform step involved. Also, you have to use some salt carrier or glycogen in order to precipitate the RNA. Are you doing this? That may increase your yield.
You cant store undisrupted tissue in Trizol, the RNA will degrade. Either homogenize it in trizol and store it or snap freeze it or store it in RNAlater - dropping a piece of tissue in trizol and NOT immediately disrupting it is a very bad thing - slow lysis and release of nucleases. That is absolutely your problem. If you are having lengthy dissections and accumulating sample, RNAlater is probably the safest way.