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What is the BEST way to extract total RNA? - (Jun/27/2005 )

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Which method gives the BEST total RNA yield from mammlain cell culture:

Quigen RNAase Easy, or Ambion PARIS, or phenol chlorofor or anyother you know of?

Thanks a TON,


Recently I did lots of RNA preps using either TriReagent and RNeasy kit plus the shred kit. I first used TriReagent in order to save some money. Although the overall results were acceptable but sometimes I couldn't get consistent amount of RNA across duplicates. This is due to human errors during RNA precipitation and washes.

The RNeasy kit gives very consistent results and takes less time, but costs more. If cost is not a problem, it is highly recommended.

The two methods are comparable in terms of DNA contamination in the RNA.


If I have many preps I'll use Qiagen's RNeasy kit. I get comparable yields with standard DTAB extraction using acidic TE-saturated phenol in the phenol:chloroform:isoamyl extraction. Do a google search and you'll come up with many DTAB protocols.



I use the Qiagen kit Rneasy. It works wonderfully! smile.gif



For speed and ease of use I think the RNeasy column method is good but you do have to careful not to overload it otherwise you will probably loose everything.

I have to use a modified Chomzinsky (Trizol) method because I am working with an infectious agent that is made safer following this protocol. I find that I usually get very good RNA but only after I run it through a Quagen RNeasy column otherwise there is phenol contamination.

RNA isolation does depend alot on the sample type so I really dont think that it is possible to categorically state that one method is better that any other. The processing by the researcher also makes a big difference... I have worked in a lab where two people were working with the same cell line and found that one person was able to get brilliant RNA out of his cell line using RNeasy while the other person got rubbish!



a question : wich ones are compatible with siRNA extraction?


I would like to add my two cents, if no one minds...

when I first started isolating RNA a couple years ago, I did parallel extractions with a sample kit from both RNeasy and Stratagene's Absolutely RNA. I did three parallel extractions (multiple samples on 3 occasions) before choosing a kit. I found the results to be better, i.e. more consistent yield, and "cleaner" RNA, from the Stratagene kit. I am thinking the prices were pretty comparable, although that was a couple years ago and there may be a bigger discrepancy now. I found that my 260/280 was on average about 1.8 with RNeasy, and about 1.95 with Absolutely RNA. The only problem I have ever had with the Stratagene kit, occasionally the 2 mL tubes that house the spin columns get warped upon closure. Otherwise, I have used it for a few thousand preps over the last two years and I get pretty consistent reliable results.

I am sure that the RNeasy kit is fine, many people use it...I always assumed it was some sort of operator-error on my part since the protocols are different. But, this is what I found when doing a direct comparison with split samples.

by the way, I use my RNA for real-time. I don't know if the preparation method can/should be different for other applications, although I don't know why it would be?


Like Haringsh, I use a somewhat similar method for RNA extraction. It is cheap, fast, and reliable (hey you can't beat that)....basically you break with AE buffered phenol in a snap freeze manner. It produces a good yield of RNA with low degredation.


QUOTE (fred_33 @ Oct 12 2005, 08:00 PM)
a question : wich ones are compatible with siRNA extraction?

silica columns eg QIAgen will not isolate anything smaller than 200nt, so they are unsuitable for this

-John Buckels-

so what protocol would you recommend for RNA that is smaller than 200nt?


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