I'm using the Ambion RNAqueous Micro Kit to extract RNA from cells in different pathways. I use laser capture microdissection to extract the cells. From one pathway, I normally collect 8-50 cells. From the other, 140-300 cells. I elute with the same amount of liquid for every extraction.
After extracting RNA, I check the concentrations on the NanoDrop One. For whatever reason, I get similar concentrations from both extractions (5-13ng/ul).
Today, I had concentrations of 11.3 ng/ul of RNA from 8 cells and 9.3 ng/ul from 144 cells.
If anybody has an idea or explanation of why this could happen I'd be very appreciative.
You may be below the accurate limit of quantitation. I see that the NanoDrop One literature does not give you values for the limit of detection or the limit for quantitation for RNA. With your samples… you may be able to detect RNA but out of the range for accurate quantitation.
Agree with the post above and I'll add you could quantify your samples more accurately with a Qubit fluorometer. There are a bunch of these in various core facilities at NIH, I bet someone in NIDA has one somewhere. These use a standard curve and are more accurate at the
Thank you guys for the answers. I see in the NanoDrop One literature (http://www.nanodrop.com/Library/3091_NanoDrop_One_UG_en_Oct_2015.pdf) that for RNA, the lower detection limit is 1.6 ng/ul with +/-1.6 ng/ul for sample concentrations between 3.2-80 ng/ul.
Do you guys still think I should go for the fluorometer?
Thanks
That will depend on your application. If you're doing any NGS application you should use a Qubit that's what most genomics core facilities do before they run samples to make sure samples are accurately concentrated. If doing qPCR it matters slightly less but at the low RNA concentration you're working with Qubit is generally better. I wouldn't pay much attention to what thermofisher or any company claims in a manual regarding limit of detection/quantification as this is almost always exaggerated.
Also, you probably know this but you can get an idea of the purity of the 9ng/µL you got with the absorbance ratios http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf
Again, all depends on what you're doing. If you're cloning or something none of this really matters, if doing NGS library prep it is very important to get the concentration right.
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