Help for a friend - molecular biology, RNA DEGRADATION - (Aug/16/2004 )
Personlly I know nothing about molecular biology but this isn't for me, my friend is studdying her phd and she is having trouble with myo-inositol phosphate synthase in yeast or something, it baffles me but this is what they say about it,
I have been having problems isolating mRNA lately. Previously, I had no problems at all. When I isolate my total RNA from S. cerevisiae the OD260/280 ratio is always good (between 1.9-2.1). I use oligo (d)T cellulose to extract poly-A RNA from the total RNA. When I measure the OD ratio for the mRNA eluted from the columns the ratio is always 1.2 or lower. This has been the case since we purchased a new stock of oligo (d)T. This has only happened one time before. The first time my advisor showed me how to isolate Total/poly-A RNA. When I redid the experiment using a new stock of Oligo (d)T it worked fine. We have since ran out of that stock and I am working with a new one. I don't know if the oligo (d)T is the problem or not. I think I am wondering what could be happening between isolating the total RNA and when I isolate the poly-A RNA that could cause the mRNA to be degraded. I do use DEPC treated tips, pipets, reagents and wear gloves. My water is always DEPC treated or nuclease free water (purchased) but I can't say how careful other students are with the nuclease free water. The only other thing I can think of is that my work area might be contaminated. Previously I used an area in the lab that no one ever walked by. Since it is now another students personal desk, I can't use it. How can I create an RNase free environment (or should I just put on a mask and a biohazard suit)?
I am assuming the RNA is degraded because when I do RT-PCR or 5' RLM-RACE I get a huge bright smear instead of a discreet band(s).
she also says it isn't the water,
thank you for any help provided,
Edited to add :
she also just said this to me, "if they ask. it isn't the reagents, tips, etc. and I do wear gloves. I have tried RNasins"
Well so much for finding help here,
Well so much for being patient...
What do you expect? I read your message, but cannot give a good hint except for "she should clean her desk with something like 'RNAse off' and be sure to use her own reagents because RNAse is kind of everywhere"?
Sometimes it takes some days until someone has an idea. The more polite you are the bigger are the chances he or she will also write it down
Greetings
CKM
Sorry it's just her teacher is getting on her back about it,
she cleans her desk with RNAse,
sorry it's just I'm worried for a friend..