Co-Purification of RNA and DNA - Has anybody any experience with the new Allprep Kit by Qiagen? (Nov/10/2006 )

Hi there,

like many of you, I always need DNA and mRNA for my methylation studies, just to check, if my favorite CpGs influence the expression of the gene.
I Have just seen that Qiagen offers a new Allprep column, which purifies RNA (like RNeasy) and DNA. Has anybody used this kit? Any experiences? Especially regarding brain tissue?

Thanks in advance,
Krümel

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can't say I have,

i use good old trizol. I would hate to know how much qiagen are asking for such a kit (a pint of my very precious blood i am sure).

Nick

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Nick,

1) is the DNA that can be extracted from trizol a high enough quality for bisulfite sequencing?
2)Do you perform any additional steps to clean up the DNA?
3) Also do you think it's okay to keep the trizol DNA phase for extended periods of time (following RNA extraction) before doing the DNA estraction?

So many questions! I only ask because alot of work done in our lab is RNA expression.....it would be nice to go back to "interesting" samples and do bisulfite sequencing on the DNA.....sometimes amount of sample is limiting.

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Ultragirl,

Yes it is, and you typically redissolve in an 8mM NaOH solution, and I go straight from there into adding the bisulfite solution for conversion


No I don't, it is usually not required, if you are careful to remove all the organic phase after ethanol precipitation and follow the sodium citrate washing.


In the notes that come with trizol, you can keep the DNA phase overnight at 4-8C and the sample pellets in 75% Ethanol at the same temperature for months. I have co-extracted RNA and DNA on the same day just in case, I haven't tried keeping the DNA phase for extended periods so I can't comment if this would work or not.

It is certainly the way to corelate DNA methylation with gene expression studies properly.

Nick

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speaking of trizol, i typically get slight DNA contamination in my RNA phase.

A quick DNAse treatment followed by cleanup does the trick.


Anyone have luck doing digests with the phenol extracted DNA?

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I've used the All Prep kit and quite frankly I'm not impressed at all. RNA and DNA purity are certainly higher off the bat. But yields appear to be much lower than from other methods, such as trizol, which can potentially be just as pure after subsequent cleanups.

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I had trouble doing digest g-DNA after trizol, tried everything I could. But the DNA works good on Bisulfite and MSP.

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Thanks Cancergeek,

that was what I had thought, too.

I am keeping up good old trizol - and it works for BSP without subsequent cleanup of DNA.

Krümel

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