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I isolated RNA form human samples with concentration ~ 500 ng/ul , 260/280 Ratio = 2 and RIN = 7.3 then mRNA isolation with concentration ~ 5 ng/ul and 260/280 =2.2 but in mRNA fragmentation step, the concentration became 20 ng/ul and 260/280 ratio more than 3, why ?
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That depends on how you quantify. Quantification in presence of salts and other reagents from buffers will less likely to be correct because most quantification assays are designed for nucleic acids resuspended in particular buffer. If you have used NanoDrop for 260/280 then you need to blank it with the same buffer that is present in your RNA reaction. -
How about posting the actual UV absorbance spectrum for your samples? Based on the peaks we see there we might be able to give you a guess as to what substance is that is confounding your concentration/prep quality assay?Originally posted by Amr Elguoshy View Post
Here is the spectrum of some of them:
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PhillipComment
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Thank you So much. I thunk that my fragmented mRNA sample may be contaminated with ethanol because UV absorbance is high at 230 nm . but How I eliminate ethanol contamination from my sample?
amr_150222_fragmented_mRNA.pdf
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Ethanol doesn't absorb UV that I am aware of. Acetate does though and sodium acetate is frequently used as a co-precipitant in ethanol precipitation. Did you fragmentation buffer have a bunch of Mg-acetate in it?Originally posted by Amr Elguoshy View PostThank you So much. I thunk that my fragmented mRNA sample may be contaminated with ethanol because UV absorbance is high at 230 nm . but How I eliminate ethanol contamination from my sample?
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Looks more like a guanidium salt though and that is not typically used in fragmentation buffers.
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PhillipComment
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