I used nano drop for checking RNA concentration of my samples and its was about 2000 ng/microliter and A260/280 was approximately 2 after that I asked myself what will happen if I tell nano drop that this sample is DNA and with this change whether I can check DNA concentrations or not, so I diid that and surprisingly the it showed DNA concentration about 6000, I checked other samples and in all of that, the concentration of DNA was about 2 or 3 times greater than RNA, So my question is whether the things I did is the right things to do or not? and if it's right to define RNA samples as DNA for nano drop for checking DNA concentrations, why the amount of DNA is so great should I change the Extraction Method, or it's sth that's usual?
Dear Motahharynia, you don't need to extract RNA again. Nanodrop is non-specific method of nucleic acids quantification. If you have to use your RNA for sequencing purpose [like transcriptome analysis], then it is highly recommended to quantify your RNA samples using Qubit RNA HS Assay Kit on a Qubit Fluorometer. But if you aim to use your RNA samples in Reverse transcriptase-PCR reactions, then you can proceed with this estimation.
For both RNA sequencing and Reverse transcriptase-PCR reactions, contamination of DNA in RNA samples is a real problem. That's why you want to make sure there is no DNA contamination in your RNA samples. But if you follow any standardized protocols, the contamination should not be a major concern.
The answer to your question is that the Nanodrop can NOT distinguish between DNA and RNA. When you tell the Nanodrop that you are measuring RNA or DNA all you are doing is changing the calculations for concentration since RNA is single stranded and DNA double stranded and they have somewhat different absorbance coefficients. But the machine can not distinguish between them. All the nanodrop can do is measure the amount of nucleic acid.
@Michael J. Benedik
Our machine for first step ask you wanna check single strand or ds.
This kind of machine can differ between RNA and DNA or not?
The RNA vs DNA setting is only to help calculate the concentrations. The peak absorbance for RNA and DNA are both at 260nm so you can not distinguish which one you have by spectrophotometry.
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