i'm trying to isolate RNA from primary osteocytes. I was able to obtain RNA samples with good purity until recently. I have tried the ethanol precipitation method with sodium acetate to purify the samples but it doesn't seem to help much.
prior to the purification step the 260/230 ratio was at 3.566. After attempting the ethanol precipitation methods, 260/230 value only dropped down to 3.388. I would greatly appreciate any suggestions on other methods that may allow to bring down the 260/230 value.
Thanks in advance! :)
Hi Thiviyakirupa,
For RNA isolation there is Trizol method and DNase treatment in addition to that to purify the samples.
About the ethanol method you used, you can prepare a new 80% to use in case it was not 80% by mistake.
Also, if your sample is very dilute then it might not measure accurately and give those big numbers. In that case the problem might not be the method but isolating not sufficient amount of RNA for that nanodrop to measure accurately.
Good luck.
One often overlooked source of variability when measuring the absorbance of RNA samples is pH; have you tried using a buffer as diluent? Take a look at this paper, it does not talk about OD230 but it might help.
https://www.ncbi.nlm.nih.gov/pubmed/9067025
Hope it helps!
Dear Thiviyakirupa,
What machine are you using for measuring the ratios? Have you exceeded the upper threshold of measuring accuracy (for example, on a nanodrop >2500 ng/ul off the top of my head).
If you have reasonably highly conc. RNA, you could try and dilute it to see what happens.
Generally though, I would not consider a high 360/230 to be a problem.
A friend of mine published a paper a few years ago comparing the efficiencies of RNA extraction kits to a cost efficient method. I highly recommend the protocol in the attached paper. I use it on a fairly regular basis. The protocol in his paper is a very quick, easy, and efficient process. I provided a link to the paper below:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5503482/
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