Hi there,

Well, use a DNase treatment first. Then check the RNA for gDNA contamination by electrophoresis, and to be 100% sure that your RNA is gDNA free you can also check if the isolated RNA amplify by PCR with some gDNA primers. If the RNA is truly gDNA free, it will not amplify by PCR.

Hope this help.

Best regards,

DNase!

Dear Rias

We usually remove gDNA during the RNA extraction using an affinity column-based kit (e.g. QIAGEN RNeasy). During the procedure, while the nucleic acids are on the membrane, DNase is added and the digested DNA fragments and the enzyme are washed before RNA elution. There are other DNase systems (e.g. Ambion DNA-free kit) that can by used after RNA purification.

And how do i get rid of dnase from the sample. Does heat inactivation work properly?

This DNAse goes with stop solution

http://www.epibio.com/enzymes/nucleases-glycosylases-dna-binding-proteins/dna-endonucleases/baseline-zero-dnase?details

I do both: add stop solution and clean with chloroform with later precipitation with EtOH.

Only chloroform/salt/EtOH should stop DNAse as well, if You want to use available DNAse in your lab without stop solution.

Ambion make some nice "off-column" DNase products. They usually come with an inactivation agent, this is added before centrifuging and then you simply pipetting out the RNA.

Hi,

Treating samples twice (successively) with DNaseI is good, too.

Hi

Treating samples with DNase and then thermal inactivation

Ria,

avoid unnecessary heats steps with RNA. RNA degrades very quickly with heat! please keep this in mind when buying kits and designing a protocol!

We've used the Qiagen product "QiaShredder" very successfully. I agree that if removing genomic DNA is mission critical, then performing this step twice is a good idea. Doing it "on-column" is also a good idea.

You can help us give you a better answer if you tell us *why* you need to get rid of all the gDNA and *how* you know you have not.

Good luck

G

Hi Grant,

Thank you for the advice. I want to perform RT-PCR for my gene of interest to check its expression level. i isolated RNA from bacteria by trizol method. I ran it on agarose gel and got the band for genomic DNA. I want to remove the gDNA for it could result in false positive results.

I used conventional "low-price" DNAseI as well as "premium products".

Both worked properly. Just try DNAseI treatment, run your qPCR and load the product from the qPCR on an agarose gel to check for the correct band.

If you have the genomic sequence and the CDS from your gene of interest you can also try to design primers on exon-exon-junctions which will make a DNAse-treatment redundant.

I normally treat my trizol extracted RNA with conventional DNase I (RNase - free grade) and heat-inactivate the enzyme. I think the thermal inactivation is sufficient for your purpose. But, if you are worried about the presence of DNase I in your sample, you can re-purify your RNA by acidic phenol:chloroform extraction or by using one of commercial RNA clean-up kits (the ones from Macherey-Nagel and Zymo Research worked well in my hands).

You can use DNase and DEPC water from Eurx

http://www.eurx.com.pl/

Hi there,

Well, use a DNase treatment first. Then check the RNA for gDNA contamination by electrophoresis, and to be 100% sure that your RNA is gDNA free you can also check if the isolated RNA amplify by PCR with some gDNA primers. If the RNA is truly gDNA free, it will not amplify by PCR.

Hope this help.

Best regards,

All advices given here are right, but you can either spend much time doing checks and controls and checks and controls of the checks and controls... ;-)

or use a conventional approach which has proven suitable by hundreds of scientists (DNAse I + heat inactivation) and keep your experiments running.

The choice is yours!

I am not able to see the 16s and 23s rrna bands after dnase treatment followed by heat inactivation.

Dear Ria,

DNase treatments works fine, but blocking it with high concentration of EDTA may reduced efficiency of the RT-PCR, so heat treatment or phenol/chloroform extraction usually preferred. another way which also works fine and specifically separates RNA from DNA is precipitation of RNA with 8M LiCl. good luck

Thank you all for the kind suggestions