I need to optimize my extraction procedure of RNA from single cell after patch-clamp recording. As I understood the first stage where i can measure the amount of extracted material - Nanodrop analysis of RNA concentration and quality. What buffer should I use in this case? Where should i put my sample, which already contains a bit of intracellular solution, RNAse inhibitor, glycogen and RNA (whole volume 1.5 µL)?
Thank you in advance!
The limit of quantification of RNA on a NanoDrop is 2 to 2.5 ng/uL. Your single cell sample, if the single cell sample contains 30 pg of total RNA in 1.5 uL, is no more than 0.02 ng/uL - which is 100X to 125X below the limit of detection of RNA on a NanoDrop. So this will be a consideration as you go through this.
If you choose to proceed, after you let the NanoDrop read a nuclease-free water sample, then, when you blank/zero the NanoDrop, you would use the solution you mentioned in your post ("a bit of intracellular solution, RNAse inhibitor, glycogen and RNA" - but adding nuclease-free water in place of the "intracellular solution and RNA" part). This would be the proper blank in this situation.
In short though, you will find it impossible to get a reading on a NanoDrop with such low-concentration RNA samples ...
you can use the elution buffer used in extraction or nuclease fee water
The limit of quantification of RNA on a NanoDrop is 2 to 2.5 ng/uL. Your single cell sample, if the single cell sample contains 30 pg of total RNA in 1.5 uL, is no more than 0.02 ng/uL - which is 100X to 125X below the limit of detection of RNA on a NanoDrop. So this will be a consideration as you go through this.
If you choose to proceed, after you let the NanoDrop read a nuclease-free water sample, then, when you blank/zero the NanoDrop, you would use the solution you mentioned in your post ("a bit of intracellular solution, RNAse inhibitor, glycogen and RNA" - but adding nuclease-free water in place of the "intracellular solution and RNA" part). This would be the proper blank in this situation.
In short though, you will find it impossible to get a reading on a NanoDrop with such low-concentration RNA samples ...
Dear Jack,
Why would you not include the intracellular solution in the proper blank? Just curious.
Regards
I would use the Bioanalyzer instead of Nanodrop for RNA quantification. Besides RNA concentration, Bioanalyzer determines the integrity of your RNA sample.
Hi Abhishek,
Due to the impossibility of actually getting just intracellular solution without nucleic acids of any kind is why water would have to suffice for that component as well. But, technically you are exactly correct: if one could actually find a way to get all cellular components without nucleic acid at all - that would indeed serve (better than just water) as a perfect part of this particular blanking solution. In fact, this kind of "matrix-only" material is lacking in all of qPCR (and other methods) in general. Anyone who finds a way to isolate [nucleic acid-free matrix-only] material from every cell and tissue type will be the CEO of a very fine company ;]
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