I have been extracting RNA via Qiagen minikit RNA extraction kit, and the conc are looking good on nanodrop (colibri) but the A260/A280 ratios are consistently shown high, in ranges of 3.6 or above. I have tried the cDNA of these RNA and its good but now we are going to sequence few samples, and are facing some issues in downstream steps, could it be a problem here in RNA purity? (it is SARS-Cov-2 RNA).
How did you know that cDNA is good? and what is type of sequencing you are going to do (Sanger or NGS)?
Pure RNA should have a 260/280 of around 2.0. So the fact that you are getting readings above 3.0 is problematic. It suggests that you have a contaminant which absorbs heavily in the 260 region. It might not necessarily be a problem depending upon what it is. However your concentration measurements are undoubtedly not correct since they are based on the 260 reading which are clearly wrong.
Might be more helpful to post pictures of your spec traces.
Phenol absorbs at 270, and phenol contamination can thus skew the 260/280.
Also, are you measuring 260/230? Because you really _should_ be (guanidium contamination).
Ahmed Elsadek Fakhr I have amplified a few samples previously (of cDNA from the RNA with such high 260/280 ratios, and have had good looking results, which could be improved, also we did Qubit based quantification of PCR product, (for Oxford Nanopore's minION), that also were average. we are doing both sanger and nanopore based WGS of SARS-CoV-2.
John Hildyard yes the instrument gives 260/230 too, and I will check them too as I have saved the results. please see this one photo from some old same type sample.
Michael J. Benedik sir, yes we have problems, but since the Qiagen is most reliable in our stock of extraction options, how can we improve it, your suggestions will be highly appreciated and helpful for us. regards
Muhammad Zakria based on that trace, I would almost certainly try to clean up the RNA. This can be achieved with a simple re-precipitation (see below), and I think that would be sufficient here: I see no 270 peak, so phenol contamination is not your issue. I suspect the source of your odd 260/280 might be free nucleotides, as supported by this thread:
https://www.researchgate.net/post/Is_there_any_difference_in_absorbance_of_dNTPs_compared_with_absorbance_of_DNA_molecules2
which suggests free nucleotides absorb significantly, and at a slightly shorter wavelength.
The bigger issue here is your 260/230, which is suggesting fairly high contamination: I typically do not use anything with a 260/230 lower than 1.7, and I aim for 2.0 if I can.
Again, re-precipitation can address this.
Add DEPC/nuclease free water to your sample to a final vol of 500ul (makes all pipetting steps easier).
Add 50ul of 3M sodium acetate, pH5.5 (0.1 vols)
Add 10ug of glycogen (optional, increases RNA recovery %)
Mix.
Add 500ul of room temp isopropanol (1 vol)
Mix.
Leave at room temp for 20mins
Spin at max speed in a benchtop, ideally at 4 deg, for 10-15 mins.
Discard supernatant (you should be able to see a small pellet)
Carefully wash pellet with ice cold 75% ethanol (~300ul)
spin again (max speed, 10mins)
repeat wash
Remove all supernatant, air dry pellet in fume hood for ~15mins
dissolve in nuclease free water (select volume based on expected yield)
My advice if you are targeting only SARS CoV2 you can use viral RNA/DNA extraction kit like Qiamp viral RNA or virus spin or Viral RNA/DNA minielute or Magnet based viral extraction kits. These kits has no phenolics and the purity will be higher provided that sample is suitable for these kits. If you need total RNA or the sample is tissue then you need RNA minikit.
Rui Chen thank you, you mean to load the RNA directly to the gel? or do some pcr?
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