Removal of DNase1
Hiranya S. Roychowdhury
hroychow at NMSU.EDU
Thu Nov 11 11:53:30 EST 1999
At 01:05 PM 11/11/99 +0000, Gary Paterson wrote:
>I need high quality RNA for macroarray analysis and so (unfortunately) A
>DNase1 step is essential to remove contaminating genomic DNA. Heat
>inactivation degrades the quality of the RNA and I'd rather not phenol
>extract bacause my starting tissue samples are very small and I need all
>the RNA I can get. Up till now, I've been using the Qiagen RNeasy kit
>in conjunction with a ribolyser which gives me good quality total RNA
>(+ some genomic) followed by DNase treatment, heat inactivation, then
>ethanol precipitation. I'd appreciate it if someone could give me
1. Following DNAseI treatment,and before heating, add EDTA to a final conc.
of about 5mM. Presence of EDTA will protect the RNA from Mg++-dependent
chemical degradation of RNA at high temperatures.
2. Precipitate the RNA away from DNA with LiCl.
3. Acid phenol extraction of RNA will also give you clean sample. The way to
avoid losing RNA from small amts of tissue samples is to do back-extractions
of the organic phase with the buffer, pooling the aq. phase together and
precipitating with 3vols of etoh. The pH of the buffer, and that of the
phenol should not be more than 5.0 to ensure the exclusion of DNA.
Sorry, I'm still not familiar with the RNA extraction "kits".
Dr. Hiranya Sankar Roychowdhury
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu
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