I am isolating a total RNA from bacterial sample for real time PCR studies. But i am facing a problem with the DNA contamination. I treated the final RNA sample with DNases enzyme. I had done a PCR with the DNase treated samples, i am getting amplification. Should i increase the incubation period of DNase treatment?
You could use the old biochemical methods, which in my opinion are always the best. LiCl precipitation will act selectively on RNA leaving DNA in solution. You might also partially lose transcripts < 150 nt long, depending also on the concentration of your RNA (but with 100 ng/ul the recovery is already very high.
The protocol is extremely simple: add to your resuspended RNA (for example after DNAse treatment) 1 volume of 5 to 10 M LiCl solution (in my experience 5M is sufficient, but you will find protocols that use up to 10 and even 12 M LiCl stock solution). Mix well and chill at -20 C for 30-60 min. Spin at 16000g for 30 min. at 4 C. The pellet is your RNA. So, discard gently the supernatant and wash briefly in 70% EtOH, Re-suspend at your convenience (I would anyway suggest, after re-suspending, to add 1/10th of 3 M NaOAc and 2.5 vol of EtOH to store your RNA as a precipitate).
Since the smaller the RNA pieces the more they are lost (do not precipitate), this treatment is not suitable for miRNA studies, but it is highly suggested for any other experiment (in vitro translation, cDNA, labelling, etc.). You will also notice that the UV quantification (Nanodrop or similar) is a lot more comparable to your gel quantification, since most of the DNA/RNA fragment/degradation products will also be lost.
You could use the old biochemical methods, which in my opinion are always the best. LiCl precipitation will act selectively on RNA leaving DNA in solution. You might also partially lose transcripts < 150 nt long, depending also on the concentration of your RNA (but with 100 ng/ul the recovery is already very high.
The protocol is extremely simple: add to your resuspended RNA (for example after DNAse treatment) 1 volume of 5 to 10 M LiCl solution (in my experience 5M is sufficient, but you will find protocols that use up to 10 and even 12 M LiCl stock solution). Mix well and chill at -20 C for 30-60 min. Spin at 16000g for 30 min. at 4 C. The pellet is your RNA. So, discard gently the supernatant and wash briefly in 70% EtOH, Re-suspend at your convenience (I would anyway suggest, after re-suspending, to add 1/10th of 3 M NaOAc and 2.5 vol of EtOH to store your RNA as a precipitate).
Since the smaller the RNA pieces the more they are lost (do not precipitate), this treatment is not suitable for miRNA studies, but it is highly suggested for any other experiment (in vitro translation, cDNA, labelling, etc.). You will also notice that the UV quantification (Nanodrop or similar) is a lot more comparable to your gel quantification, since most of the DNA/RNA fragment/degradation products will also be lost.
Which protocol do you use?
you can use Fermentase instruction. Its included 1000 ng of your RNA sample, 1 ul of 10X reaction buffer with MgCl2 ,1 ul of DNase I in a final reaction volume of 10 ul.
After that, incubate for 30 min at 37 oC
inactive the enzyme by adding 1 ul of 50 mM EDTA solution to the reaction mixture
incubate for 10 min at 65 oC.
The protocol complete, you can do a PCR. Good luck.
I followed the same protocol of Fermentas for DNase treatment. But still got amplification in the conventional PCR method using RNA sample.
Dear Ganga are you sure about your enzyme? maybe it doesnt work.
Could your DNase enzyme ineffective or some error during experimentation. I am following below protocol for DNase treatment.
http://tools.lifetechnologies.com/content/sfs/manuals/18068015.pdf
Add DNAse to your preparation and see how much time it takes to inactivate all DNA molecules, and set that timing for incubation.
The only DNase enzyme that I trust, after extensively testing them on a sample with plasmid "contamination", is the Turbo DNA-free (Ambion, now LifeTech). Follow the protocol carefully. Trying to treat RNA at above the recommended concentrations or amounts decreases the efficiency of the reaction.
I have no experience with the biochemical methods but I would recommend testing that the precipitation doesn't affect the subsequent PCR since qPCR enzymes can be more sensitive to salts than regular Taq.
Just if you are curious, Sherri, Li+, contrary to the other mono or di-valent cations, is not precipitating with the nucleic acid, even if you use it in combination with EtOH in place of Na+ or NH4+, and it is recommended also by Ambion (Life Technologies) for RT-qPCR.
Thanks all for your valuable information. Finally sort out the problem. Had problem with the enzyme. I had changed the enzyme and now its working ...
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