It seems that loss of RNA is unavoidable when it comes to DNAse treatment, but I seem to be losing ~3/4 of my RNA. I quantify my RNA & DNA amounts with Qubit (and NanoDrop RNA yields are similar). During (in-column) DNAse I treatment my RNA-concentration drop e.g. from ~280 to 60 ng/µl, whereas my DNA-concentration drops from ~10 to ~2 ng/µl. The RNA is going to be sequenced. I'm using Direct-zol RNA MiniPrep (Zymo Research), and extracting RNA from Drosophila flies.
Any suggestions how I could save more of the RNA?
Unfortunately, a large number of commercial DNase preparations will also degrade RNA (due to aforementioned RNase contamination etc), in particular when applied on-column. Lexogen has documented this in the SPLIT RNA Extraction Kit Application Note (http://www.lexogen.com/fileadmin/uploads/pdfs/SPLIT/008.048_SPLIT_AN_008AN019V0100_2014-08-27.pdf)
In the development of the SPLIT kit, we have optimized gDNA removal by a combination of classic acidic phenol extraction and silica purification, without the need for extra DNase treatment. If - e.g. from RT-qPCR or agarose gel evidence - you know that DNase treatment is still needed, do not perform it on-column but in solution and do not heat inactivate but remove the enzyme by either phenol extraction and/or silica purification.
You will find more information on these topics in the SPLIT User Guide, accessible at http://www.lexogen.com/products/split-rna-extraction/split-downloads.html
This seems quiet strange to me because DNAse I cannot digest RNAs even in case the RNA form DNA:RNA hybrids.
The reason for your loss should be researched in other processes, maybe RNAse contaminations of your DNAse I reagents?
I agree with Paolo, maybe you could use any sort of RNA protector. I think there are many commercial solution for that.
Unfortunately, a large number of commercial DNase preparations will also degrade RNA (due to aforementioned RNase contamination etc), in particular when applied on-column. Lexogen has documented this in the SPLIT RNA Extraction Kit Application Note (http://www.lexogen.com/fileadmin/uploads/pdfs/SPLIT/008.048_SPLIT_AN_008AN019V0100_2014-08-27.pdf)
In the development of the SPLIT kit, we have optimized gDNA removal by a combination of classic acidic phenol extraction and silica purification, without the need for extra DNase treatment. If - e.g. from RT-qPCR or agarose gel evidence - you know that DNase treatment is still needed, do not perform it on-column but in solution and do not heat inactivate but remove the enzyme by either phenol extraction and/or silica purification.
You will find more information on these topics in the SPLIT User Guide, accessible at http://www.lexogen.com/products/split-rna-extraction/split-downloads.html
I also had problems with on column digest and solved it by using the Ambion DNA Free Kit which works in the Eppi quite fast and no precipitation needed afterwards...
I have a lot of experience with RNA isolation for microarray analysis. My samples consisted of very minute amounts of cells, making excellent RNA recovery very important. Like Norbert, I am a big fan of Ambion's Turbo DNAfree kit which not only provides a quick and easy DNAse treatment after column purification but a column-free resin-based way to remove the DNAase with minimal loss of RNA. As a bonus, the resin (which is proprietary but I assume has an antibody for DNAase on it) also chelates some cations that can destabilize RNA if present when RNA sample heated to higher temperatures (> 55C) for downstream reactions. Hope that helps.
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