RNA Isolation- Help Needed!

[DallasRNA]

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

Isopropanol precipitation

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!

[Stefan-CoA]

Are you using RNase AWAY? Could be just residual RNase floating about (I hear they're quite persistent). I know that in the lab where I worked as an undergraduate they used that stuff at every opportunity. Spraying gloves and work surfaces and equipment with it.

[DallasRNA]

Yes, we use RNase ZAP from Ambion, sprayed on everything from gloves to surfaces. Using RNase free tubes, tips, etc. But even if the RNA was degrading, wouldn't I still get an [RNA] out of the Nanodrop and it would just fail an integrity assay? I have aqueous phase backup from the TRIzol in my freezer but I don't want to use it up if I am just going to get results like this!

[CharonY]

Well, reproducible RNA extraction is often somewhat tricky (hence all the normalization steps). I have not used cartilage, by my feel is that the reproducibility of initial lysis is the major factor here. The low cell to crap (i.e. ECM) ratio makes it hard to have precisely the cell amount, as well as inhibit complete homogenization.