Trizol extraction - (Jul/11/2008 )

Hi,

I've been trying to extract RNA from macrophages for an microarray study using different kits, but end up with unacceptably low RNA yields. The only method that gives acceptable yields is Trizol extraction, but I am worried about DNA contamination issues.
Has anyone got experience with the use of Trizol-extracted RNA for microarrays? What are the recommended purification steps?

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After that you could use the purification kit of either Invitrogen or Qiagen product, just follow the manufactruer's instruction.

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Trizol usually gives good yields -around at least 600ng/ul and then purify it further with the invitrogen/qiagen/promega kit. That should work..

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Hi,

I am working with Microarray experiments and have experience in RNa extraction by Trizol method. You can do a Qiagen Column clean-up with Dnase treatment after the trizol method. This will remove DNA contamination.

Cheers,
Teena

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I use this method with TRI reagent.... give me good yield and clean RNA.

http://www.path.cam.ac.uk/~toxo/Protocols/...20ISOLATION.pdf

regarding the genomics DNA contamination issue, we find that problems lies in the types of tissue you use.
In my case, i just purified with QIAgen column and no DNA contamination found. But one of my friend who use different types of tissue has serious problem of genomic DNA contamination in her sample. She MUST use DNase to treat with her sample during purification.

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I have been using Trizol/ TRI reagent for RNA isolation for microarrays.. In my experience, Trizol RNA is fine for microarray experiments if u take good care not to carry over any of the interphase from the Trizol and give another chloroform extraction followed by 2 ethanol washes

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What we do is adding another phenol:chloroform step (using low pH phenol), and LiCl clean up.

Sometime we don't even need DNase our RNA.

Hope this help

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OR using BCP solution instead of phenol: chloroform for better separation.
if you scare of DNA contamination, put more trizol/tri-reagent, then be more careful with taking the upper phase.

trizol/ tri-reagent always give high yield... why do you say it is low? You determine by using nanodrop or bioanalyzer?
if your sample is too concentrate, you'll get funny when determine using nanodrop or bioanalyzer, coz the concentration exceed their limit.

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I think you should try DNAse treatment try to extract the total RNA from fresh cell lines or else if you are handling any tissues you can store the tisues at -2o but for extraction you should always do homozination and than give Trizol treatment after extraction you can give DNase treatment for labelling atleast 15-20 ug of total RNA is required.

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TRIZOL is light sensitive and is often stored in a dark-colored, glass container covered in foil. It must be kept below room temperature. When used, it looks exactly as if it were cough syrup, bright pink. The smell, more specifically, the smell of the phenol and guanidinium thiocyanate, are extremely strong. TRIZOL works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components.
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