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RNA extraction for qPCR: Trizol vs. column?


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5 replies to this topic

#1 BottegaVeneta

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Posted 07 October 2009 - 11:55 AM

Hi,

I've been having trouble getting clean RNA with Trizol. I try to do everything carefully (ie: not touch the interphase after chloroform extraction, etc). I also resuspend my final RNA in water, but I heard you can resuspend RNA in TE buffer and get better A260/280 readings. Is using a column better than the more time-consuming Trizol method?

Thanks!

#2 fishdoc

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Posted 07 October 2009 - 12:10 PM

Hi,

I've been having trouble getting clean RNA with Trizol. I try to do everything carefully (ie: not touch the interphase after chloroform extraction, etc). I also resuspend my final RNA in water, but I heard you can resuspend RNA in TE buffer and get better A260/280 readings. Is using a column better than the more time-consuming Trizol method?

Thanks!



I use the RNeasy kit for bacterial RNA. It works great. I had tried using Trizol a few times, but it never worked all that well. The RNeasy kit takes 30-60 mins and always gives a good yield.

#3 BottegaVeneta

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Posted 07 October 2009 - 03:35 PM

I wish there was a cheaper alternative that's just as quick and easy as a kit. I have to do RNA time courses, and the price really adds up. ;)

#4 littleaxt

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Posted 12 October 2009 - 01:42 AM

Hi!

I always had problems with gDNA contaminants when using the colums, so I changed to Qiazol (more or less the same as Trizol) and got much better reults. but in the end I apply both no, Extraction with Qiazol and after DNase I digestion I make a clean-up with RNeasy columns. I get really pure, high quality RNA but it is also very expensive :-(

#5 Johannes

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Posted 12 October 2009 - 05:02 AM

in case of miRNA a lot of recent papers describe better yield using TRIzol than any available kit.
we are using TRIzol for RNA extraction and qRT-PCR on miRNAS, too. No Problems so far.

#6 moerae

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Posted 26 October 2009 - 10:28 PM

We use both Trizol and columns in our lab.... Kinda an overkill but RNA is really clean afterwards.




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