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about the total RNA isolation (trizol methods) and further Dnase I - (May/12/2009 )

about the total RNA isolation (trizol methods) and further Dnase I inactivation methods

I just start to work on the miRNA qRT-PCR fields and try to establish some assay, the method is firstly to add poly (A) at the 3-end of miRNA, then use SYBR green to perform qPCR, I am still confused about some points:

1.After the isolation of total RNA (Trizol methods from invitrogen), Dnase I treatment is required (promega kits), then to stop the Dnase, I need add 50mM EDTA to stop the reaction and 75 C 10min incubation the enzyme.

But, the next step I use poly (A) tailing kits to add poly (A) at the 3-end of the miRNA, in the kits manual (ambion), it said EDTA should not be added to stop the Dnase I reaction, or it will affect the poly A tailing and further RT-PCR reaction.

How to handle the problem? Are there any other methods to stop the Dnase I reaction except EDTA? Or should I purify after stopping the Dnase I reaction by phenol-chloroform? If so, I will have to extract and purify RNA two times by phenol-chloroform since usually I will purify RNA after poly A tailing added before to reverse transcripase the first cDNA strand.

2. In trizol method to isolate total RNA, do you use scraper to harvest the cells? and do you use 1ml syringer and 0.8mm needle to homogenize the harvest cells?

Thanks a lot in advance.

veers

-veer2-

Is DNase treatment necessary for miRNA isolation? Another purification just to get rid of EDTA is unacceptable. The ambion kit should provide a solution to that conflicting problem. Take a look at Ambion's website, this page provides several alternatives of inactivating DNase http://www.ambion.com/techlib/tn/83/836.html

After adding trizol, scraping is not necessary, you can use a 1000 ul pipettor to pipet the viscous lysate up and down until it is homogeneous. Adding adequate trizol can minimize DNA contamination.

-pcrman-

really thank you very much for your kind assistance.

I have another question: After the trizol isolating total RNA, usually how much microgram total RNA do you start the Poly A taling and reverse transcription? Usually for usually reverse transcription, 2ug total RNA is enough, but for miRNA, how much dou you start? 20ug? or even more?

Thanks a gain

veer

-veer2-

veer2 on May 13 2009, 09:49 AM said:

really thank you very much for your kind assistance.

I have another question: After the trizol isolating total RNA, usually how much microgram total RNA do you start the Poly A taling and reverse transcription? Usually for usually reverse transcription, 2ug total RNA is enough, but for miRNA, how much dou you start? 20ug? or even more?

Thanks a gain

veer



SEE this paper:
MicroRNA isolation and stability in stored RNA samples.

Mraz M, Malinova K, Mayer J, Pospisilova S.

Biochem Biophys Res Commun. 2009 Dec 4;390(1):1-4. Epub 2009 Sep 19. Review.

-Gaetanodiitaly-