Need to improve RNA integrity - RNA Isolation (Jan/06/2005 )
After following the Invitrogen method for RNA isolation form small quantities of tissue, I extracted 132.8mcg/ml of RNA. However, my 260:280 ratio was only 1.4955. I need to improve this number to about 6.0 or 7.0. Does anyone have any suggestions?
Do you mean 1.6 or 1.7?! 260:280 of 6.0 would be absolutely amazing. I routinely use Tri-Reagent for extraction and suffer the same problem. I ignore it for the most case when using tissue culture pellets, but when I extract from animal tissues I often re-extract one or two times with phenol: acid-chloroform. Ambion is a good source for such reagents and information on working with RNA. (www.ambion.com)
The alternative is to pruchase RNeasy columns from Qiagen. I use them when preparing RNA for microarray and the yield puritiy ratios >1.9. You could always take your existing extractions and pass them though the columns, I believe there is a protocol in the QIagen manual for doing this.
The Biology Ninja
I agree with the ninja. I personally like the RNeasy kits because I consistently get RNA with a 260:280 around 1.9 and you can somewhat control the concentration based upon how many times you run water through the column (the last step of the protocol). Also, I am not a big fan of working with phenol/chloroform and the RNeasy uses columns as an alternative. Good luck.
Of course...if you follow the Qiagen protocol, you have to add B-mercaptoethanol to the lysis buffer. BME stinks like poo poo.
I called Qiagen tech support to see if I could omit the BME. They tell me it is neccessary for denaturing RNases. Unless I'm mistaken, BME reduces di-sulfide bonds largely. I don't know about it's ability to denature protein. They tell me I cannot substitute DTT for the BME...I don't know if I believe them but have yet to be adventureous enough to attempt using DTT in lieu. Anyone tried something besides BME?
DTT reminds me of popcorn. It's yummier to smell.
- The Biology Ninja