I am trying to isolate RNA from vesicles but despite the RNA concentration by spectrophotometry. I am concerned by the 260/230 ratio of these samples, all values around 0. What could be a reason for that?
For note, EVs were purified by sequential centrifugation. Then, the vesicular RNA was isolated using a commercial kit.
Thanks in advance!
Dear Alexis,
Although you did not mentioned the type of extraction kit used in the sample preparation and extraction, this very low ratio is due to contamination of your RNA with guanidinium isothiocyanate and/or phenol as well as the presence of some proteins. The most effect on your RNA comes from the guanidine isothiocyanate. All these three factors are either present in your extracted sample or are present in most commercial lysis solutions such as in Qiagen kits,….etc. In your case it may be due as well to very low specimen RNA content or combination of all aforementioned factors. I would also suggest do the following:
1-Increase the initial volume or concentration of the preloaded sample to the extraction column. Do not over load the column with larger amounts as this will overload the RNA column or beads.
2-Be sure that you stabilized your RNA in the sample from RNAses` degradation. 3-Try an extra column wash step to remove residual salts by using slightly longer and slightly faster centrifugation steps at room temperature.
4-Note that sometimes the lower pH of the purified RNA solution affect the absorbance reading at spectrophotometer.
You can try use Qubit RNA Assay Kit for the sample RNA extraction.
"good luck"
Dear Najat Abdulrazzaq Hasan ,
Thank you for your answer and suggestions. Surely, it will help in my next experiments.
About the method used for isolation, it was the miRNeasy serum/plasma kit (Qiagen).
RLT, which is a buffer used in the Qiagen RNeasy RNA isolation kits also contains a guanidine isothiocyanate-like component that may interfere significantly with RNA OD measurements. The RLT give large absorbance below 230 nm.FTI.
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