Hi Maccus Hao Jie Wong

DNase treatment is clearly the best way to rid an RNA sample of contaminating DNA. However, some preparations of DNase may be contaminated with RNases, and the DNase must be completely inactivated prior to RT-PCR so that it doesn't degrade newly synthesized DNA. Unfortunately, removal or inactivation of this enzyme is problematic; DNase removal methods can be inconvenient, ineffective and even detrimental to RNA integrity.

Kindly look at the protocol in link.

http://fg.cns.utexas.edu/fg/protocol__DNAse_treatment_of_RNA.html

This protocol addresses the issue of genomic DNA contamination in RNA samples using the Ambion (Applied Biosystems) TURBO DNase. We want to remove DNA contamination to the best of our abilities because our interest in environmental stresses is in the gene products that arise from exposure to a stress condition. Since we convert the RNA back into DNA via reverse transcriptase, and then attempt to amplify target genes that we believe to be responsible for the cell’s stress response, any genomic DNA present could also be amplified, leading us to question whether the final results of RT-PCR are due to the cDNA that we generated, or simply the genomic DNA contaminating our sample. To guard against this, we treat RNA samples with DNase, an enzyme that selectively degrades DNA.

Good Luck

RNA contamination can be evacuated by including 2 microlitre of RNase A (10 mg/ml, Fermentas) to 20 microlitre of DNA broke down in TE cradle (Tris– EDTA, pH = 8.0) and hatch for 3– 4 h at 37 C.

If you want to get rid of your RNase you can can phenol:chloroform extract your DNA after the RNase digestion.

regards

Retreat your samples with RNaseA and then purify using Phenol chloroform isoamylacetate ph 8 . Additionally you could use a PCR product cleanup kit like one from Qiagen and run your samples through columns.

Hi Maccus Hao Jie Wong

DNase treatment is clearly the best way to rid an RNA sample of contaminating DNA. However, some preparations of DNase may be contaminated with RNases, and the DNase must be completely inactivated prior to RT-PCR so that it doesn't degrade newly synthesized DNA. Unfortunately, removal or inactivation of this enzyme is problematic; DNase removal methods can be inconvenient, ineffective and even detrimental to RNA integrity.

Kindly look at the protocol in link.

http://fg.cns.utexas.edu/fg/protocol__DNAse_treatment_of_RNA.html

This protocol addresses the issue of genomic DNA contamination in RNA samples using the Ambion (Applied Biosystems) TURBO DNase. We want to remove DNA contamination to the best of our abilities because our interest in environmental stresses is in the gene products that arise from exposure to a stress condition. Since we convert the RNA back into DNA via reverse transcriptase, and then attempt to amplify target genes that we believe to be responsible for the cell’s stress response, any genomic DNA present could also be amplified, leading us to question whether the final results of RT-PCR are due to the cDNA that we generated, or simply the genomic DNA contaminating our sample. To guard against this, we treat RNA samples with DNase, an enzyme that selectively degrades DNA.

Good Luck

Hi Maccus Hao Jie Wong

First, you would be wiser to omit the DNAse step because it just leaves you with short pieces of DNA which, as you have discovered, are often hard to eliminate. There are two approaches to separate RNA and DNA, as you are trying to do. One is to do a salt precipitation step where you dissolve the nucleic acid to a concentrated level (and make sure the DNA is fully dissolved) then add NaCl to give 1M final (or LiCl to 1M final), mix well and place on ice or at 4C. Under these conditions only RNAs (in fact only large RNAs, so including the 26/28S and 18S along with the mRNAs) will precipitate, DNA will stay in solution. If the RNA is concentrated enough the solution will become cloudy fairly quickly after being placed on ice, and you should leave the tube for at least 15 minutes but probably 30 to 60 minutes is better. Spin the tube and the pellet should be pure RNA; if you had a lot of DNA contamination then you might have to repeat the salt precipitation.

The second way to eliminate the DNA is to dissolve the nucleic acids and then add NaCl or sodium acetate to 0.3M final, mix, add 2 to 3 volumes of 95/100% ethanol, mix well and you should see the DNA snap out of solution fairly quickly as the mass of threads you expect for DNA. Have a thin glass rod or a Pasteur pipet ready with the end having been bent (in a Bunsen flame) into a small hook, and use that to grab the DNA out of the tube. The RNA will of course precipitate eventually, but it does so more slowly than DNA and it also has a granular or particulate form which will not come out with the DNA mass (except possibly for a little RNA simply trapped in the DNA thread mass.)

DNase treatment, by its very nature of being an enzymatic process, is affected by the efficiency of the reaction. So even in a reaction that is 99% efficient, 1% will remain. However, my work shows that DNase treatment can be very effective with the efficiency of the DNase treatment being more than sufficient for downstream RT-qPCR applications. Furthermore, the Alu PCR metric is very sensitive for detecting gDNA presence and has shown DNA levels remaining at never more than 1 in 5 million copies of RNA with an effective reduction of gDNA of up to 99% (unpublished data).

Alu PCR reference: Vermeulen J, De Preter K, Lefever S et al. (2011) Measurable impact of RNA quality on gene expression results from quantitative PCR. Nucleic Acids Res 39 (9):e63

In terms of the size of DNA fragments remaining post-DNase treatment, so long as the fragments are smaller than the amplicon size, then this remaining DNA will have no impact on a RT-qPCR result. Furthermore, larger DNA fragments would need to represent the specific amplicon region in order to influence an RT-qPCR result. Given the likely small quantity of such fragments, any influence on the RT-qPCR result will be negligible. Other downstream application such as RNA-seq may be affected by small DNA fragments.

The difficulty with performing multiple clean-up procedures is that you will reduce your RNA yield, which will particularly impact your ability to measure low abundance targets.

Both approaches are valid depending on the sample used and the downstream application.

Hi Issam Jumaa Al-Ani and Mushtak T.S. Al-Quqaili

Thanks for the very long answer, unfortunately i am afraid that the answers are irrelevance with my questions. My questions are focusing on the RNA contamination not DNA contamination. Thanks for the efforts of putting the answers together.