How many microglial cells do I need to be able to do qPCR?

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Hidetoshi Tozaki-saitoh

I sorted 3000 of CD11b(APC conj.)positive cells from spinal cord by FACSAriaIII.

RNA was extracted using a popular kit for small size sample.

Usually beta actin was detected (Taqman) by qPCR(roche lightcycler) at 26 - 30 cycles.

Harald Lund

Thanks Hidetoshi! I'm impressed - that's very few cells. Have you been able to run other genes for example inflammation-related genes? And what was the kit that you used to extract RNA? Thanks again.

Hi.

I used "NucleoSpin RNA XS (MACHEREY-NAGEL)". I will try "Quick-RNA™ MicroPrep (ZYMO)" next. Since it does not seem to contain carrier RNA.

I could detect chemokines( CCL2,3,5), TNF alpha and IL1 beta (it needs 35-45 PCRcycles). Presence or absence was clear in several inducible inflammatory genes, but I think more cells are needed for exact quantification.

Elisa York

Hi Harald,

I am doing a similar protocol now (FACS isolation of microglia for RNA isolation and qPCR). I'll be using the RNeasy mini kit.

How did it go for you? What kind of cell numbers did you get, and how much RNA does that translate into?

Hi Elisa!

I have only now gotten around to doing these experiments, and I am struggling with low yields of RNA (using Rneasy kits). Have you gained any valuable knowledge since you asked your question?

Pooja Shree Mishra

Hidetoshi Tozaki-saitoh,

Isnt 35-45 cycles a doubtful scenario, esp if your use sybr green? i have got good yield with astrocytes , and an IL6 expression at about 28-30 cycles. However extracting rNA from microglia is really, really tough.. so far with t-25 flasks, i havent been able to get beyond, say, 60ng/ul with RNAeasy kits. fairly low!

How many nano grams of CDNA did you use for a 20/40 ul reaction? i use 1ng/ul of reaction for astrocytes, that works fairly well. No idea with microglia though.

Thanks.