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I am currently conducting an experiment which involves FACS-sorting a specific population of cells using an antibody of interest.

In order to validate the type of cells I have collected using this marker, I want to perform qPCR on their extracted RNA, using certain known cell-type marker primers.

My problem is that in each sorting session, I am only able to collect anywhere from 5,000 - 10,000 cells. Do you know if it is possible to perform RT-qPCR on such a small sample size?

I have collected the cells in 1mL of Trizol.

Would I maybe have to amplify the cDNA after reverse transcription in a general manner ( using random primers)?

Thanks for your help.

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  • $\begingroup$ Wouldn't hurt to try. With the right primers and enough PCR cycles you should be able to amplify just about anything, maybe not perfectly though. $\endgroup$ – user137 Jul 20 '15 at 4:58
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    $\begingroup$ I think this should be possible, although you will not get very much cDNA, so you probably have only one shot. Besides this: Why are you using so much Trizol, I typically used 1ml for one well of a six well plate, which has ways more cells. When you precipitate, make sure your are using some co-precipitation agent as glycogen, so you won't lose sample. And you will probably not see the RNA pellet with so few cells. $\endgroup$ – Chris Jul 20 '15 at 5:32
  • $\begingroup$ Thanks Chris. How much Trizol would you recommend? I'm using 1mL because I sort into 1.5 Epp tubes and try to prevent the cells from sticking to the walls of the tube when they exit the sorter. Would amplifying all the cDNA after RT make sense? (Non-specific, just random primer amplification?) $\endgroup$ – gaelgarcia Jul 20 '15 at 6:01
  • $\begingroup$ Aren't you going to amplify the cDNA in your qPCR anyway? A pre-amplification would probably cause trouble with your data. $\endgroup$ – user137 Jul 20 '15 at 7:26
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    $\begingroup$ 1ml trizol is quite a lot. I use that much for ~1 million cells (a well of a 12-well plate- for HeLa. You generally need half of that). You can also try one of the RNA extraction kits. Add DNAse-I to remove any traces of DNA. Don't do a random primer amplification. Take a look here $\endgroup$ – WYSIWYG Jul 20 '15 at 13:18
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You can consider use of single-cell RT-qPCR kits such as this one if you have the budget for them. (Note: I have not personally used this kit, just putting out an idea)

The Ambion® Single Cell-to-CT™ Kit contains a complete validated workflow for gene expression analysis for samples containing 1–10 cells. Each kit contains reagents for sample preparation, reverse transcription, pre-amplification, and qPCR that have been optimized together in a simple workflow that can be completed in only 5 steps (see figure).

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With good technique, you can reliably perform RT-PCR or RT-qPCR on a sample size of about 100 cells. Some of the comments are correct that you will need to compensate for the reduction of the initial cell input with additional PCR cycles. However, each magnitude in reduction of cells corresponds to about 3 extra PCR cycles (2^3 = 8 ~= 10), so your overall qPCR cycle number will not change significantly.

Since you are dealing with a small amount of cells, and isolating by TRIzol, the pellet could be difficult to see. I would suggest you use something like Glycogen (Glycoblue from Ambion) to co-precipitate the pellet into something more visible. You would add this prior to the Isopropanol to precipitate your nucleic acids.

Your use of extra TRIzol is not a problem unless you're lab is short of money. It will dilute your concentration of nucleic acid, which will make precipitation harder, but using glycogen will compensate for this. Using an amount less than the manual directed ratios can be problematic as it could result in incomplete lysis and denaturation of your cellular components.

As user137 recommends, Pre-amplification of the products is not a good idea as it will likely distort your final results. I'm not sure what gene expressions you're looking at, but it is always a good idea to include multiple housekeeping genes as baselines to calculate your relative expression numbers, provided you have enough RNA isolate to use as input.

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