I am extracting RNA using a Zymo Research Direct-Zol RNA Miniprep kit. I've tried using their DNAse I in-column as described in the manual, but I have been ending up with a substantial amount of gDNA contamination after reverse transcription. Has anyone else used this DNAse successfully, or should I give up and do DNAse treatment in solution after purification?
Hi, I have the same problem. I figured this out as my RNA preps give signal in qPCR without cDNA synthesis (some housekeeping genes are detected with probes that do not span intron). Furthermore, the in column DNase reaction did not significantly alter the presence of gDNA. Zymo Research says I could do the reaction in solution as well, but then I should use an extra column to purify the treated RNA.
I've always had better results doing DNase I treatment in solution rather than on columns. I would recommend using the DNA-free kit from Ambion, as the inactivation reagent works extremely well without requiring re-purification or precipitation of the RNA for downstream applications.
Best of luck with your project!
Hi, I have the same problem. I figured this out as my RNA preps give signal in qPCR without cDNA synthesis (some housekeeping genes are detected with probes that do not span intron). Furthermore, the in column DNase reaction did not significantly alter the presence of gDNA. Zymo Research says I could do the reaction in solution as well, but then I should use an extra column to purify the treated RNA.
I came to the conclusion that the in-column treatment is entirely useless and ended up doing a separate DNAse treatment. Thanks for the guidance!
The same problem is giving me trouble. I started to use the Direct-ZOL column two weeks ago with cultured cells. Nano-drop showed good quality of the RNA samples, however, qRT-PCR gave me terrible results. The CT valune of actin usually is 18, but I got 28 with Direct-ZOL method. I had thought that the problem probably came from the lower cell number and repeated again with more cells and got same results. I agree that the in-column gDNA digestion does not work well. I am thinking about adding chloroform to get the supernatant and then use the column to avoid the gDNA contamination.
I've been using Direc-zol for plant samples. Although the concentration and ratios are very good I suspect there is some polyphenols in my sample as the gels has a smear near the well and my primers ( previously working) does not detect any cDNA.
In my case I let the DNAse for 30 minutes in the column and I don't have problems with gDNA.
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