I use Qiagen RNeasy kits and do use DNase (15 minutes treatment) but still end up getting gDNA contamination. I prepare RNA from about 3 million N2a(neuroblastoma) cells line. I further want to analyze the gene expression through qPCR and hence a pure RNA isolation without gDNA contamination is extremely important.
Hello Pearl! I'm using the same kit as well for my E.histolytica cells but I also apply extra DNase treatment by using the Invitrogen turbo DNA-free kit after elution step. Another possibility is, your DNase might be out of date.
hello Pearl Cherry. you can treat RNA with different Concentrations of DNase like 1U/ug of RNA, 2U/ug. and also can extend DNase Treatment for further 15 mins at 37'C. hope you will get DNA free RNA.
- Agree with Irem
- you can use different concentration of DNase for different time interval at different temperature
Hi all,
Thanks for all the suggestions highlighting the use of DNase or turbo. What if I tell you that there is a technology which is based a unique "Carbon-Surface Chemistry" and is specifically designed to just isolate RNA with no detectable DNA contamination by PCR, would you like to dive into that? Oh! and this technology is backed up by National Science Foundation innovative grants and doesn't relay on any DNase treatments. Send me an email at [email protected] and I can walk you through some more details.
Hi Pearl Cherry , I am facing the same problem. Which brand of Dnase were you using? I extracted the RNA using RNeasy Mini Kit and performed Qiagen Dnase on column digestion. I still have gDNA contamination in my qpcr NRT samples.
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