Reading RNA ODs again.. - RNA extraction (Dec/10/2007 )
Dear All,
I have extracted RNA using the Trizol method but have obtained quite low OD (260/280) for some samples ranging from 1.5-1.7. I read somewhere that before reading ODs its a good idea to heat the RNA at 65C for 10 mins. I was wondering.. do you think it's possible for me to maybe do this step after I have read the ODs already and have stored the RNA at -80C? Will the thawing and heating to 65C affect the RNA in any way? Im just thinking that perhaps it might give me a better OD reading if I do this step..
Thank you all for your help!
L
I would say, go for it, try it and you will find out. I heart from other people that for a strange reason (strange to me) storing your sample overnight at -80°C, then measuring it gives more reliable data then immidiatly measuring the sample.
Possible explanation why heating up can help is that proteins precipitate at higher temps. But the procipitated proteins are still in your sample
Thank you so much! I'll definitely give it a go..
I really appreciate your reply 
Thanks again,
Laura
I really appreciate your reply
Thanks again,
Laura
huh??
is there any difference btw heating and no heating ??
i isolate my RNA from yeast.
i ever try heating to 65C before i measure the OD, and also try to measure the OD straightly after i dilute the RNA in water..
I always thought the heating step was done to destroy secondary structures: 
I have extracted RNA using the Trizol method but have obtained quite low OD (260/280) for some samples ranging from 1.5-1.7. I read somewhere that before reading ODs its a good idea to heat the RNA at 65C for 10 mins. I was wondering.. do you think it's possible for me to maybe do this step after I have read the ODs already and have stored the RNA at -80C? Will the thawing and heating to 65C affect the RNA in any way? Im just thinking that perhaps it might give me a better OD reading if I do this step..
Thank you all for your help!
L
What are you diluting your RNA in? Eg: If you dilute in water you'll get a lower ratio than with TE buffer (for total RNA). What will you be using the RNA for? It might be ok for downstream applications - I would always spec my RNA and use it even if the ratio was on the low side.
hi all
im gonna tell you my opinion and how i work. hope not to confuse you more.
first i always work in low tempratures. i never let my RNA get hot. i believe that is a very delicate molecule and if the temprature goes up there might be degradation going on.
also i believe, because of the nature of the molecule, is generally very difficult to manipulate it. I never get the same ODs doing exactly the same things.
before my ODs count, i prewarm (i leave it ON) the photometer at the desired wave lengh for 30min. then i count ODs.
I always run a 1ul sample of my RNA in 1% agarose TBE DEPC EtBr gel and check out if what im seeing on the gel confirms my OD counts.
hope helped a little bit...
t