RNA Extaction from microglial cells culture - the best method for RNA extraction of microglial cells culture (Nov/14/2008 )

Hello,

I've recently started to set up a microglial cell culture in my lab. We do it with neonatal rat brains and the number of cells that we obtain is not too big, but I think that its enough for RNA extraction and isolstion and for RT-PCR techniques.

I usually employ Trizol method to extract RNA from my cultures and it works quite good for neurons and astrocytes but when I do it with microglial cells the quantity of RNA that I get is too poor and I cant get the minimal quantity required for cDNA transcription, that in our protocol lab is of 100ng in 10 ul. Does anybody know if these cells need of an specific RNA isolation protocol? I've heard about adding glycogen to the trizol in order to improve the concentration of RNA in the chloroforme phase, could it really significantly improve the quantity of RNA that I have? and finally, I dont really know what to do :-), I see a white phase in the tubes, that should be the genomic DNA, so I think I have a good number of cells, but at the end, there is not RNA. Could the phenotype of my cells (small and rounded) be implicated in the extraction?

Thanks for your help

Kind regards

I would like to send you a sample of AquaRNA to try out. You could use it directly (without using the toxic phenol/chloroform) or add a volume to your trizol and go as usual (increasing yield 2-3x (see attached gel) and stability).