RNA spectrophotometry - (Mar/21/2006 )

Hi,

What sort of ranges could you expect for usable RNA (I want to do RT) in the spectrophotometer, in terms of 260/280 and 260/230 ratios?

I am getting high readings for 260/230, and I am not sure if I should just do a new extraction?

thanks

search tool of bioforum please !!

http://www.protocol-online.org/forums/inde...topic=11738&hl=
http://www.protocol-online.org/forums/inde...807&#entry13807
http://www.protocol-online.org/forums/inde...?showtopic=5968

Thanks Fred,

Those links explain a lot about the 260/280 ratio.

I would also like to know about the 260/230 ratio. What is the optimum? What is too high/low to use?

thanks

Good quality RNA will have an OD 260/280 ratio of 1.8 to 2 and an OD 260/230 of 1.8 or greater. This is because nucleic acid is detected at 260 nm, whereas protein, salt and solvents are detected at 230 and 280 nm. A high OD 260/280 and OD 260/230 ratio therefore indicates that you have extracted RNA devoid of any of these contaminants.

Vetticus

ok, I did another search and found this old thread

http://www.protocol-online.org/forums/inde...?showtopic=6412

in which it says that the 260/230 should be higher than 2 if its uncontaminated. Right?

In that case, it seems that there is something wrong with our Agilent Bioanalyser or with one of the reagents in our kit.

I got a very strange result with the Bioanalyser. I thought this might be due to solvent contamination in the RNA, since it looked ok on the gel.
But on the Spectro, my 260/230 readings were all above 2.

Hmmm. Has anyone else had strange (broad humps instead of peaks) results on a Bioanalyser, when their RNA was actually ok?

hi
well i forgot to add links for 260/230.
You're right. Ratio should be more than 2. I get usually more than 2.5

I would suggest sending the data file to Agilent as soon as possible. They will diagnose far quicker than we can

(I was at the user meeting yeterday and they kept saying "send us your files, send us your files")

Good idea, I will do. Thanks all!