Ajay: could you kindly explain how DEPC treated water/plasticwares are enough to "get rid of DNA contamination"? It is interesting that you are using DEPC treatment for last 5 years to get rid of DNA.

Abdolmajid: autoclaving will not destroy DNA detectability by later PCR-based assays.

Milos: you have not spelled out how you treated your RNA sample with what DNase prep, how you purified the RNA afterwards, and how you measure your RNA before and after DNase treatment. Knowing these details will be important in addressing your problem. Also, do tell about your RNA species. Is it heterogeneous as extracted from cells/tissues or is a homogeneous species as derived from in vitro transcription?

Dont use DNAse in that case dear Milos. Infact if you are using properly DNAse/RNAse free plasticware and avoiding contamination, there is no need of using DNAse. We are doing the procedure since last 5 years and never used DNAse, instead use proper DEPC treated plasticware and dissove the RNA in DEPC water. That is enough to get rid of RNA contaminations.

Ajay: could you kindly explain how DEPC treated water/plasticwares are enough to "get rid of DNA contamination"? It is interesting that you are using DEPC treatment for last 5 years to get rid of DNA.

Abdolmajid: autoclaving will not destroy DNA detectability by later PCR-based assays.

Milos: you have not spelled out how you treated your RNA sample with what DNase prep, how you purified the RNA afterwards, and how you measure your RNA before and after DNase treatment. Knowing these details will be important in addressing your problem. Also, do tell about your RNA species. Is it heterogeneous as extracted from cells/tissues or is a homogeneous species as derived from in vitro transcription?

First, I want to thank you all for kind replies.

Second Ajay, as Anil already stated, I hope that you made a unintentional mistake when you stated that you rid of DNA contaminants by free plastic ware and DEPC water! These precautions that you mentioned really help to decrease activity of RNAse and to not damage your RNA sample, but DNA contamination comes from your sample when your are isolating your RNA, not from outer contaminants or similar.  These precautions should be applied to decrease potential RNAse activity as I stated.

Anil: I agree with you, that is. I am using rat prefrontal cortex, I isolate RNA using TRIzol reagent and dissolve it in DEPC water. Afterword, I treat my RNA sample with DNAse I kit to get rid of genomic DNA and reverse transcription to get DNA from RNA (well  checked protocols by Fermentas and by us).  After DNAse treatment, I dont check my RNAse and add master mix for reverse transcription. When we stared we checked our RNA after DNAse treatment on agarose gel and measured RNA concentration on nano drop and we got very good results. Namely, our RNA was a very good quality (integrity, two bands on gel), we get rid of genomic DNA contamination and good RNA concentration (indeed lower that without DNAse treatment, but still quite well). We run today three different RNA samples with and without DNAse treatment and we were surprised when we found 5 fold lower RNA conc and degradation of our RNA, smear on agarose gel.  

That is worrisome, Milos! Is this recent DNase of the same lot as the previous ones? If yes, how long has this lot been in use, and how many people have access to this kit? I hate to say this, but inadvertent and unintentional errors can often contaminate enzymes. As you know, the smear in your gel indicates degradation. And, yes, it can also happen with frequent thawing. So, find a way to restrict access of your RNA to too many colleagues; make aliquots and store in -80. Besides, can you have some EDTA in your DEPC-treated water that you use to dissolve your RNA (and please maintain your OWN custody of this water and make sure that you are the only one who uses it)? I would suggest you get a new lot. Fermentas kit should be just fine; I use the Ambion kit though! Alternatively, you could also try Sigma Tri-Reagent (http://www.sigmaaldrich.com/technical-documents/protocols/biology/tri-reagent.html); it claims that it does not bring in DNA contamination. Indeed, I have done RTqPCR from my RNA with or without Ambion DNase treatment with similar amplification, indicating that the manufacturer's claim is correct. However, I use this reagent to isolate RNA from in vitro transcription reactions. Your experimental need is different.

Being a heterogenous RNA population has its advantage. It may so happen that there is no RNase contamination, and your transcript of interest may be more labile than the others. So, to test this possibility, you could pick 2-3 genes randomly and see if their levels change proportionately -/+ DNase. Your recent gel, though, does not give confidence to me!

Please update your newer finding; that will help others in similar problems. Good luck.

@ Anil and Milos, yeah dear that was an unintentional typing error for DNA instead of RNA (corrected the same). Milos, what I am trying to say that if you are talking about inherent DNA contam then try not to take the viscous phase along with the transparent RNA (upper phase). Sometimes, we are very greedy about quantity of our samples but in that case you should focus on quality as kits are available which can easily detect even fg of RNA and succesfully make DNA. Have a look if u r making this mistake or not.. Best Of Luck !

Hi Milo, Going through all the replies.

I think, as Ajay mentioned, relying only on Specto quantification is not right. You should try doing RT-PCR for IC gene  and check the left over quality of RNA after DNAse treatmen..

Secondly as Anil mentioned, You should categorisely keep your stocks of reagents separately, DNASe should be prepared in DEPC treated water and all glass and plastic wares should be made or being used should be RNASE free (DEPC treated and baked at high tolerable temp).

How ever, I have one query, What shd be the conc of DNAse 1 if I am using a powder of   DNAse 1? and How to make it (in DNAse Buffer OR DEPC Water?)