Handling small quantities of RNA - tips? - spec quantification of ~50ng? (Jul/27/2006 )
I was wondering if anyone here has any tips on how to handle small quantities of RNA without losing RNA, i.e. how should I quantify my RNA if I only have less than ~50ng? It seems like a catch22 situation - I don't want to have a high dilution factor because the spec readings will be so low and probably unreliable, yet I don't want to have lower dilutions and waste my valuable RNA!
I've heard of people using a "nanodrop", but I don't think that we have one, and it's extremely unlikely that we would buy one just for quantification of a few samples.
Any tips would be most welcome!
Nanodrops are ideal for low quantity samples. Agilent Bioanalyzer is another way to do it if you have access to one, and they can give you visual and quantitative idea of RNA integrity.
What you may want to do use is a fluorescent assay to estimate concentration. I'm familiar with the Quant-It RNA sysem from Molecular Probes. You can compare your samples against a standard curve for concentration, its pretty accurate and very sensitive. You will need access to a fluorometer. A plate reader that can detect flourescence is ideal with limited samples.
You might also use the Hoechst 33258 quantitation assay. I'd first go with a NanoDrop though.
Load 0.5ul RNA sample on EB agrose gel plate, and compare with a serials diluted RNA standard (0.5ul each)
Thanks for the tips. We do have an Agilent Bioanalyser, so I guess that will be the best method if it is able to give an indication of the RNA integrity.
great! You should only need about a ul and you'll get much more info than a spec read.