poor RNA quality - (Aug/31/2005 )

I am trying to isolate RNA for real-time RTPCR...I snap freeze my brain tissue upon collection because I must perform a micro-dissection to obtain my nuclei of interest. I use Trizol purification methods and my quality is poor. Among other things, I am wondering if the fact that the cryostat used for dissection of my tissue is not sterile (I do not have an RNA only cryo) is a problem?

I'd say that would be a big problem... RNAses are everywhere and they have a nasty tendency to chew up your RNA before you get to work with it. Something that's used to cut up tissue will be completely contaminated with them.

Maybe you could try and get some RNAseZap from Ambion to clean the blades before using them?

The other thing you could try is to use your cryosections in a specialised RNA extraction kit. I don't have any personal knowledge how that would work, but those kits generally guarantee reasonably good quality.

LeserattePD

Hi,

I isolate RNA from brain tissue too and I am using Roche High Pure RNA Tissue Kit.It is ok for me :)It contains all the reagents that you will need.Only thing that you should be careful about is to incubate your RNA for more than 15 min. with DNase I (supplied )for example for 45 min. otherwise it does not work

Hope it helps.see you...

Thanks to both of you! It is a big help. I have some RNase zap and will try cleaning and putting in a new blade- I can't believe that never occurred to me. Also,I have used the High Pure RNA kit and been unsuccessful. Maybe I will try the 45 min incubation you suggested.
Thanks again!

Hi,

I isolate RNA from brain tissue too and I am using Roche High Pure RNA Tissue Kit.It is ok for me :)It contains all the reagents that you will need.Only thing that you should be careful about is to incubate your RNA for more than 15 min. with DNase I (supplied )for example for 45 min. otherwise it does not work

Hope it helps.see you...

The fact my RNA is dirty (260/280)=1.4 will influence a lot my rtPCR?

Thanks


Luca,