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Old 07-15-2015, 02:06 PM   #1
PhatB
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Default Pooling FACS sorted single cells to increase yield - Best method to concentrate RNA?

Hey all,

I am attempting to pool RNA from single cells that have been FACS sorted in an attempt to increase my RNA concentration before cDNA amplification and library prep. The reason I have to pool instead of sorting more cells/well is that I have to determine if the RNA in each well contains viral RNA or not. I will then pool cells with or without viral RNA. The issue is that if I start pooling RNA samples in lysis buffer my lysis buffer concentration will quickly surpass the maximum amount required for Clontech's first-strand cDNA step. What would be an effective way to concentrate the RNA without losing it or degrading it?

Cheers!
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Old 07-16-2015, 05:00 PM   #2
kerplunk412
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It sounds like you are planning to extract the RNA from the single cells first so that you can check for the viral RNA, and then you would like to pool and concentrate the RNA from multiple single cells. If this is correct, I think a good old-fashioned ethanol precipitation may be your best bet, as you could probably resuspend the RNA directly in the lysis buffer. You may want to use linear polyacrylimide as a carrier, as I believe there are some reports of residual mussel rRNA in glycogen. Which is too bad, because I think glycogen makes a much nicer pellet.
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Old 07-16-2015, 06:14 PM   #3
HESmith
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Glycogen is a known inhibitor of reverse transcriptase (LPA is inert). But it does make a nicer pellet...
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Old 07-16-2015, 09:13 PM   #4
PhatB
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Maybe the pooling method would be too much handling and lead to potential degradation? The core facility at my research institute uses Clonetech's ultralow kit that contains a 10x reaction buffer for cell lysis prior to cDNA generation. The problem is that it would be too cost prohibitive to sort into this buffer since if I sort 100 cells in the reaction buffer in separate wells but only use 15 samples that contain the virus then I wasted a bunch of reaction buffer. I don't know if they sell it separately.
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Old 11-23-2015, 03:07 PM   #5
efrainrib
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check out the kits by Zymo

I pool facs sorted nuclei directly into trizol LS out of the machine. Then i use the zymo direct-zol kit and get highly concentrated RNA in a min of 12ul, which is crucial becuase the clontech kit as you described only allows for 8ul.

i have also used the clean and concentrator -5 from zymo as well. works great. this sounds like its exactly what you need. just run a pilot and see how little you can elute in without leaving RNA ont he column. i.e. spin it once with 8ul then put in another 8ul and see if you can extract any more RNA on the second run. then you can figure out what your minimum volume would be
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