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Question about RNA extraction & gDNA elimination


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#1 Bunsen Honeydew

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Posted 28 March 2011 - 09:11 AM

Hi there,

I use the RNeasy kits from Qiagen for my RNA extractions. I follow the protocol, however normally perform my gDNA elimination step once I have eluted my RNA. I then DNAse digest using a kit from Invitrogen (Life Technologies) as per manufacturers protocol. I then reverse transcribe my RNA for qPCR.

I wondered how many of us perform the on-column digestion step as recommended by Qiagen? If so, how do you rate your RNA quality (I only have access to a Nanodrop & sadly not a Bioanalyzer) and your yield? Do you prefer on-column or post-elution, do you think it makes any difference? Does anyone mix and match other manufacturers, eg RNeasy on-column digestion with Invitrogen DNase?

Has anyone tried the new RNeasy kit which has a special gDNA eliminator column (I'm slightly dubious about this as I'm working on a rare cell population and wonder how much RNA I'll loose running things though another column...)?

Does anyone perform a purification/clean-up step following DNAse digestion?

Any suggestions/questions/comments gratefully received :)

Thanks,
Bunsen

Edited by Bunsen Honeydew, 28 March 2011 - 09:20 AM.


#2 Nephrite

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Posted 29 March 2011 - 09:25 AM

Hi :-)

About the philosophy of protocol I can`t say anything because I am not experienced and I learn this techniques now. I can share what I have been doing so far.

I work with RNAs from paraffin sections. For RNA extraction I use Qiagen RNeasy FFPE kit, Cat No 74404. So far I followed strictly the instructions but yesterday I increased the incubation time with proteinase K from recommended 15 min to 30 min. Everything is the same as in the protocol. I just loaded 1 microgr of my RNAs on 1.2% gel - I don`t see gDNA. From these RNAs I am usually able to generate cDNA fragments with length ~350-400bp, using random primers. So, I think that gDNA elimination should not be a problem.

Nephrite.




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