I have extracted RNA from avian blood using TRIzol from ambion. After a 3M sodium acetate / ethanol precipitation, the RIN number reads >8 on the bioanalyzer. I want to treat the RNA with TURBO DNase (ambion) after the TRIzol extraction. To inactivate the DNase before RT PCR, would it be best to re-extract the RNA from the solution using TRIzol, chloroform and isopropanol?
I would strongly recommend re-purifying after DNase, but either with SPRI beads or a column kit. Bits of leftover trizol or phenol or chloroform can interfere with RT.
Hi Sarah, thank you for your answer. I've tried using the qiagen micro kit dnase digestion and then using the min elite columns for clean up. It looked quite good on the bio analyzer, although the RIN number was below 8 (~7.4). My 260/230 ratio on nano drop was also low after this.
I was hoping the turbo dnase might be more effective, but maybe I just need to do a sodium acetate precipitation after the kit clean up?
The degradation is likely coming from well before the DNase step. If you really want higher RIN values then I'd try a different purification method - one of the Blood specific clean up kits. A RIN in the 7's is common for tissue extracted RNA.
Hi Sarah, how necessary is a RIN number of greater than 8 for a qpcr? I am looking at gene expression using plates coated with primer for 84 genes involved in the immune response
For qPCR lower RIN's should be fine as long as you do the proper control genes etc. If they are >7 it should be ok. I'd be cautions even with qPCR with
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