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.
Edited by veer2, 12 May 2009 - 12:12 PM.