RNA Isolation - RNA isolation from human cell lines (Aug/25/2009 )
First post and time on one of these forums so please patient and i hope you can help.
I have human tracheal epithelial cell lines, i grow up on 24 well plates, take off and use Qiagen RNasy kit to produced pure RNA.
I have done this a couple of times now but still getting low 260/230 value. Everything else seems fine.
I have added in an extra step of buffer RPE and also i have added in a step of leaving the caps open on columns for 5 min to evap off any EtOH and also tried heating up RNA free water for last step.
These suggestions i got from google and did help with 260/230 value which is now up to around 1.5 - 1.8 but this is still a bit low. DO u have any suggestions for this?
Also i am not sure what the graph should look like on the nanodrop for pure RNA.
I think it should look this.
However my graphs sometimes dont dip at 230 nm, more at 240nm (i still have a peak at 260) is this a problem? is this due to my 260/230 ratio being low?
Sometimes i get a peak at 230 and nothing at 260 what does this graph tell me?
Thanks for any help, i would like to be able to read more into the graphs, as i know what all the ratios mean.
Welcome to the forum.
low 260:230 is due to precipitated salts and possibly phenolics. I suspect that the problem is with the Qiagen kit, I have seen other similar posts from users of this kit. Try dissolving the RNA in 10 mM Tris pH 7.5, precipitate with isopropanol and re-suspend in tris again.
I will give this a try.
Also have you any advice on reading the graphs produced?
i.e. for RNA isolation is it good to have a dip at 230 and a peak at 260? if so what do these dips and peaks mean?