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

Ann Sutherland via methods%40net.bio.net (by as9n from virginia.edu)
Thu Sep 17 16:30:37 EST 2009


>Hi Adam,

I have routinely done RT-PCR on samples of 100 mouse embryos at the 
8-cell to blastocyst stage (so 800 to 6,000 cells total) with a wide 
variety of primers.  I think you may not be quantitatively recovering 
your RNA in step 2.  For large samples, Trizol works really well, but 
for small samples I think the percentage lost is too high.  Instead 
of Trizol, I used the Micro-Fast Track kit from Invitrogen, and later 
the micro PolyA+ kit from Ambion to isolate polyA+ RNA from small 
samples and got great results.  I also routinely use glycogen to 
co-precipitate with the RNA to get better yield.
Ann

>------------------------------
>
>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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>End of Methods Digest, Vol 52, Issue 11
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-- 
Ann Sutherland
Associate Professor
Department of Cell Biology
University of Virginia Health System
1300 Jefferson Park Ave.
Jordan Hall 3-15
Charlottesville, VA  22908

ph:  434-243-6711
FAX:  434-982-3912
email:  as9n from virginia.edu



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