miRNA isolation and detection - (Jul/02/2008 )

Hi everyone,
I am new in the field of miRNA and have some questions.
I want to isolate miRNA from blood cells (PBMCs) and want to know whether anybody has experience with that.
What isolation method would be a good choice?

To then test whether the isolation was succesfull I want to detect miRNAs by an ABI TaqMan assay.
Is there any specific miRNA which should be used for this kind of cells (PBMCs) and what kind of control should I use?

Are there any miRNAs that are expressed abundandly in all cell types?

Thanks alot

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To your first query, check some of these links: http://search.vadlo.com/b/q?sn=158621799&a...blood&rel=0

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Recently we used Qiagen miRNeasy kits to extract total RNA including miRNAs, it worked well.

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I've already worked with PBMC. just TRIZOL or any TRI reagent that you have in your lab. It's easy, straightforward and quick. result is a lot of clean total RNA containing miRNAs. that's it.
I don't know about specific miRNAs for PBMC, but mir16 is surely highly expressed. Taqman probes from applied biosystem work very well.
hope it helps

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Thanks, that helps alot.
Could you give me your Trizol Protocol?
Just addition of Trizol, then resuspend the cells (or also an incubation step at 15-30°C), addition of chloroform, shake, centrifuge, and precipitation of total RNA by isoprop, wash with EtOH and dry?

Did you count the number of PBMCs? And have you ever tried to store the PBMCs in RNAlater at -80°C and then isolate total RNA?

Many thanks

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You just have to follow the normal protocol as it is, no changes needed, your total RNA will contain also the miRNA fraction. I tried with 200.000 cells and it worked just fine. with more cells is a lot easier!!!
You can store PBMC in trizol at -80°C for at least 1 month (even more in my experience) without problems.
Can't say about RNAlater, i don't use it, sorry.
If you need anything else write a PM, i'm quicker in answering.
Bye
Fizban

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