We are conducting a measurement of Ribonucleic acid from baker's yeast (S. cerevisiae) (Sigma-Aldrich R6750) concentration using Eon Biotek Microplate reader. We dilute the RNA in various concentration (ranging from 100 microgram/mL until 1000 microgram/mL) using deionized water. Then we move the samples to a 96 UV-Vis plate and calculate the absorbance of the samples using Eon Biotek Microplate reader in lamda 260 nm and 280 nm. The results are quite confusing, first get the trend by the increase of concentration, the absorbance also increase, but then when we tried to calculate the RNA concentration using:
[concentration] = 40 x Absorbance 260 nm x dilution factor
we cannot get the right result,. Then when we calculate the A260/A280 ratio, the values are below 1, which can be an indication that there is too much protein in our samples.
My question is where the problem come from? Is it from our procedure, the purity of the RNA, or the setting of our microplate reader?
Hi there,
As you are working with commercial stuff I would tend to think there is something wrong with your plate reader calibration. I would suggest to establish the full UV spectrum using a usual cuvette spec or a nanodrop to have a look at the overall shape of the absorption spectrum. Did you also pass a miniprep plasmid DNA sample in your reader to see what 260/280 ratio can be obtained with a reference sample?
Quality requirements vary depending on the downstream application. RNA sequencing and microarray experiments require high quality RNA samples with specific purity and integrity (A260/A280 bigger than 1.7 and RNA integrity number (RIN) bigger than 8.0). In contrast, for qPCRbased assays and northern blot analysis, samples with lower quality scores may be used. I advise you to re-isolate your RNA samples. If the 260/280 ratio is lower than 1.7 this means there are impurities on your RNA sample (like residual phenol, guanidine and proteins). For more information, you can read this article
Article Isolation of High‐Quality RNA from Pichia pastoris
I think Dominique Liger has a great suggestion, try to measure one or two of your samples using a traditional spec and quartz cuvette or something comparable. Another thing to check is to confirm that the plates you are using are actually suitable in the 260nm range. Many UV-Vis plates only work satisfactorily down to 300 or 320 nm, you need special plates designed for use in the range below 300nm.
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