RNA Isolation Help!
Posted by: mfrank
Date: April 29, 2011 11:36PM
I am isolating RNA from cartilage, which is notoriously difficult due to the low cell count and stiffness of the tissue. The eventual application is q-PCR analysis. We use a hybrid TRIzol/RNeasy protocol that goes something like this:
homogenize with trizol,
three extractions with TRIzol/BCP
Resuspend in 150uL RNAse free H20, mix with equal volume 80% EtOH
Apply to QIAgen RNeasy spin column and clean according to directions.
Elute in volume of 30 uL, running eluate through membrane twice to increase RNA yield
On a good day, average yield is 30-40 ng/uL, with a 260/280 of 1.8+, and 260/230 of 1.3 ish.
On a great day, can get as much as 70ng/uL, with slightly better readings of 260280 and 260/230.
But sometimes my results are all over the place! Yesterday, I did fifteen isolations, and two of them were very good in yield and ratios. In the rest, though, I had a yield of only 5-10ng/uL, an awful 260/280 ratios of 1.2-1.5, and 260/230 ratios of 0.3-0.8.
I just don't get it. Why, on the same isolation, am I somehow losing my RNA in many of my samples? I had lovely pellets for all of them, saw them dissolve in the water before applying to the column, and somehow through the qiagen protocol I lost everything? Does anyone have any clue? We are not overloading the column since there is a capactiy of 200ug, and even at my best 70ng/ul, that is only 2 ug in a total of 30uL!
Please help, it is so frustrating and I don't want to lose all of my samples!