How are you testing for DNA contamination? Intron spanning PCR?  Any chance you could have isolated some nascent RNA?

Actually we sent our RNA samples for quality check by Bioanalyser and its showing a extra sharp peak along with 18s RNA and 28s RNA. so we can't move to  next step required for RNA sequencing.

How are you testing DNA contmaination? With wich buffer are you incubating your sample with DNase? Have you tested it against some DNA to see how it stands? You could alternatively buy a RNA isolation kit, like Direct Zol from Zymo. It comes with a Dnase.

I also use the Zymo Column kit and incubate with DNAse I for 15 min. Make sure you're not loading too much material: try to stick to less than 1 ug. QC bioanalyzer should look good. 

We have our best luck when we dilute the RNA down to 200 ng/uL or slightly under, then DNAse it. Maybe this is compatible with your needed downstream use? We do this twice in a row, with a different DNAse than came with the RNA purification kit (so, in our case, enzyme from Ambion). We also were suggested by tech support to use 1.5X the DNAse buffer (so instead of 5 uL of their 10X buffer, we used 7.5 uL in a 50 uL reaction). This all helped. Good luck!

Hello, I used to use Dnase to kill the DNA in my RNA samples. I incubate my samples with Dnase1 for 10 minutes without shaking or pipetting, then I deactivate that by warming my samples in waterbath at 70 degree.

If you are using phenol/chloroform method, try to do the phenol:chloroform:isoamyl not to reach pH 8. This will favor more RNA extraction than DNA. later you may further purify using DNAse as mentioned by Dr. Rana Al-Baghdadi.

One thing that has bugged me since your first reply is that you said you were getting a 'sharp peak' along with the 18/28s RNA.  I've never used a bioanalyser so refrained from answering but  If this was genomic contamination would it not appear as a smear?  If it's a sharp band the DNA must be of a restricted range of sizes (unless of course it so late in the run that size cannot be discriminated) so how, apart from the bioanalyser, have you verified that it is DNA that your seeing? 

Dear Trivedi, additional peaks in Agilent Analyzer are either due to RNA degradation or fragmentation, due to gDNA contamination and often by technical / mechanical errors. All these peaks differ in their heights, widths and locations on electropherogram (as these peaks could be in the pre-region, post-region, in inter-region or in the fast region). Comparison of these digital peaks with the gel image is very important to reach the final conclusion about any additional peak identity. Mechanical spikes are mostly in post region and due to micro bubbles or tiny particles introduced into the chip. If your 28s and 18s peaks intensities and heights are good and no jagged peaks or multiple peaks especially in the pre-region and in fast region and gel shows no multiple diffused bands at lower region so your RNA looks fine. gDNA, due to its heavy weight appears usually in the post region of electropherogram, due to large molecular weight its peak is quite broader and your gel image will be fuzzy or highly smeary above the 28s rRNA band, so likely it is the gDNA, but if peaks other than 18s and 28s are found in other regions with bizarre appearances so you must rule out the issues of RNA integrity first. In gDNA case usually the treatment is using DNAse and heat denature your samples at 72 C for 2-3 minutes to get rid off all the possible secondary structures. So final decision about any extra peak individuality is based upon the Agilent electropherogram and gel image both.

SDS-acid phenol method is a best method to isolate DNA free RNA samples.

I highly recommend TURBO DNA-free kit from Ambion.

I tested the activity by various concentration of plasmid/RNA mixes and really never detected plasmids by qPCR as far as I tested.

@ Yutaro Hori ....Thanks! Actually we have ordered this kit so will try once we recieve it.