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RNA isolation from subcutaneous white fat


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#1 sammowammo

sammowammo

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Posted 14 March 2011 - 07:27 AM

Hi all,

I'm having the most difficult time isolating RNA from subcutaneous fat. However, I don't have a problem isolating RNA from other white fat depots. A weird thing I've noticed when I use Trizol for phase separation is that the layers are pink on top, followed by interphase, and clear on the bottom. This occurs for both epidydimal white fat and subcutaneous white fat. In a previous experiment, I took both pink and clear layers from epidydimal white fat, continued the rest of my RNA isolation protocol, and measured RNA concentration. I found that the "RNA" from the pink layer displayed a higher RNA concentration and an optimal 260/280 ratio, was able to reverse transcribe into cDNA, and yielded good C(t) values for 18S when I ran the cDNA on qPCR. So, this whole time, I have just been taking the top layer. A lab mate of mine says she takes the top layer too even if it's pink because this frequently occurs when isolating white fat. But this doesn't happen all the time, and I'm trying to determine what is wrong with this sucker (subcutaneous fat). I ran a gel on the RNA I isolated from subcutaneous fat and it wasn't there (in all of my precious samples!). We don't have RNA mini columns that are specific for isolating RNA from fat (by Qiagen), but we have an RNA mini kit from Invitrogen. I might try that out. And I might also use more Trizol and extract with chloroform twice instead of once. If you guys have any ideas, please let me know. A lab at UVA does the same thing, and they only use RNA columns when doing microarrays. But other than that, he says Trizol should work. Help please!!

Thanks,
Sam




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