qRT-PCR on very few (10,000 - 20,000 cells)

Adam . via methods%40net.bio.net (by anonwums1 from gmail.com)
Wed Sep 16 20:13:58 EST 2009


Hi all,

I am attempting to do qRT-PCR using material from 10,000 - 20,000 cells.
Here is what I'm doing:

1) FACS sort 10,000 - 20,000 cells into Trizol LS
2) Prepare RNA in Trizol LS per standard protocols
3) Make cDNA from the RNA using Invitrogen's SuperScript III First-Strand
Synthesis System for RT-PCR
4) Perform qPCR on that cDNA using Applied Biosystem's RNA-to-Ct kit using
Sybr green as a detector

I have verified using 100,000 cells that I can amplify my target product.
The problem is that when I go down to my actual samples of 10,000 cells,
nothing amplifies, not even my housekeeping gene. I've seen people publish
qRT-PCR on rare cell populations, but they rarely tell you how many cells
they actually had to collect to get any usable data. Unfortunately for me,
those 10,000 cells requires 5-10 transgenic mice and 5 hours of prep time,
so simply increasing the cell number is probably not viable.

Any suggestions on how to fix this? Do any of you routinely amplify such
small amounts of RNA for qPCR or qRT-PCR? Do you amplify your RNA first
using commercially available kits. If so, what kits?

Thanks,
Adam


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