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

[time pedal]

Hi everybody,

Which qPCR machine is the best choice in your opinions?  Need to buy one but
not sure what to look at first.  Many thanks.

T

On Thu, Sep 17, 2009 at 2:30 PM, Ann Sutherland <as9n@...> wrote:

> 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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>
>
> --
> Ann Sutherland
> Associate Professor
> Department of Cell Biology
> University of Virginia Health System
> 1300 Jefferson Park Ave.
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