2 replies to this topic

[maria777]

Hi, I usually isolate RNA using the Trizol method. I homogenize for 30 sec the mouse pancreas in 1 ml of Trizol, then I follow the phenol/chloroform protocol, always working in ice. The quality and the amount of RNA are good (i.e. 6 mg/ml ratio 1.8), but when I perform real time PCR I am always able to see the GAPDH expression and nothing else. If GAPDH values are around 25-30 and each other gene (lipase, amylase, elastase) are undetermined or around 35-38, is it possible that I don't see the expression of other genes because GAPDH is too low? When I have isolated RNA from pancreatic acinar cells (where no homogenization is necessary) with the same protocol, the quality is similar, the amount is 0.6 mg/ml and after real time PCR GAPDH is around 20 and the gene of interest is there at 25-28. Is it possible to avoid homogenize the tissue? How should I clean the homogenizer?
Should I use proteinase K? If yes, when and how?

Thanks for your help.

[markusda]

Did you check your RNA on a gel ???? (or better a bioanalyser or microChip) ? Maybe you have some contamination in your preparation which inhibitis your reverse transcriptase.

pancreas is reach in Rnases, try a qiagen RNA-kit.

To decontaminate the homogenizer, use 0.2 M NaOH overnight, and clean carefully with DEPC-water....

[Carlton H]

Maria,

If your homogenizer may be contaminating your samples, allow me to suggest one of our products that I think can help you.  Our Bullet Blender can homogenize tissue while it is inside of standard microcentrifuge tubes, so the homogenizer never needs to contact the sample and you can run up to 24 samples at a time with none of them ever coming in contact.  Check it out.  If there's any questions you have or anything else I can help you with, be sure to let me know!

Also, and forgive me if you've heard it all before 100 times, but the most common problem in anything involving RNA is RNAse and humans practically ooze the stuff.  Be VERY careful about your lab technique - ALWAYS wear gloves, and even going so far as to wear a face mask can't hurt (or, if you don't, make sure not to breathe on or talk in the direction of your samples).  Also, when some of my lab was doing lots of in-situs, we would have some bench space cordoned off as "RNAse-free" that we only used for RNA work.  All of the above can't hurt.

Cheers,
-Carlton