Hi guys.
I make RNA by in vitro transcription and do cell transfection afterwards.
I analyze RNA using Nanodrop and Bioanalyzer.
But I found the concentration of RNA measured by these two machines are significantly different.
For example, I got 300 and 303 ng/ul by Nanodrop, but I got around 800 and 600ng/ul by bioanalyzer, using the same sample.
It seems that results obtained by Nanodrop don't change and results by bioanalyzer change a lot.
Which one should I trust? Because I have to calculate using the concentration for cell transfection.
Thank you!!
Hello Ka Tou
The Nano Drop is meant to quantify nuclei acids by simply measuring absorbance at 260nm and 280nm. It does not give a perfect reading and so it is not reliable especially for those samples which are not pure.
On the other hand, the bioanalyzer uses fluorometric dyes that intercalate with RNA and with the help of the RNA chip it detects RNA. This instrument is not used for quantitative analysis. It is used to evaluate the integrity of RNA sample by providing the RIN score.
For accurate quantification of RNA I suggest you use the Qubit Assay kit and the Qubit fluorometer for measuring the fluorescence.
Regards.
I agree with Malcom that a Qubit is a better measure than the Bioanlyzer. However, as long as you have purified your in vitro transcription product to remove free nucleotides and the DNA template, the Nanodrop should be plenty accurate for measuring concentration.
I've used spectrophotometers to quantify the IVT mRNA that I've used in my in vitro and in vivo experiments with no issues. I'd recommend using the same instrument to measure each time regardless of which you choose.
I'd also recommend analyzing your IVT products by gel electrophoresis to confirm the product size (do this again if you polyadenylate the RNA to check the tail size too). You can also do a relative comparison of different samples if you run them on the same gel.
Hi there,
I would be more confident in Nanodrop because as already mentioned it relies on direct absorbance reading whereas Bioanalyzer requires fluorescence signal calibration if you intend to use it for quantification.
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