I have isolated RNA from bacterial cells. But I want to be sure that there is no contamination with DNA. How can I be sure? Which experiment is suitable for that?
Hi Khan,
as very well suggested already, you should always do a DNAse treatment after RNA extraction. Nevertheless, a PCR reaction on your gene of interest or any RNA you can think of (including ribosomal RNA) without a reverse transcription reaction (so, directly on your RNA) will have to give negative results, since DNA polymerases cannot use RNA as a template. If you amplify something you still have some DNA around. It is good practice in any RT-PCR experiment (quantitative or not) to run a RT- sample.
It would depend how much you know about the genome of your bacterium, but an easy way would be to try to amplify, by PCR, a non-transcribed region of the genome. If you get amplification you have some DNA contamination.
Hi Khan
It is better to treat your samples with DNAse and since bacteria doesn't have introns-exon organization in gene structure you need to be sure of that for many subsequent experiments....Do a -RT PCR control to see te elevl of contamination.
bye
Hi Khan,
as very well suggested already, you should always do a DNAse treatment after RNA extraction. Nevertheless, a PCR reaction on your gene of interest or any RNA you can think of (including ribosomal RNA) without a reverse transcription reaction (so, directly on your RNA) will have to give negative results, since DNA polymerases cannot use RNA as a template. If you amplify something you still have some DNA around. It is good practice in any RT-PCR experiment (quantitative or not) to run a RT- sample.
Hi Khan
If you really want to avoid DNA contamination try to use one step quantifast kit this will convert the purified RNA to cDNA and run the amplification in one tube reaction. best of luck
you could go for the absorbance ratio . A260/A280 if it comes around1.8 means you have DNA cotamination in your RNA extraction and if it is above 2.0 means you RNA is pure..for this we use nano drop...
in our lab, we first check the A260/A280 ratio, and then after cDNA synthesis reaction, we check some genes expression using primer can amplify the fragment including intro, if there is DNA contamination, 2 bands can be detected, one is come from cDNA, and other longer band is come from DNA contamination. And please treat your sample with DNAse.
Definitively nanodrop is not going to solve your problems. I always check DNA contaminations by PCR, using the same primers and thermal cycles that I will use in the RT-PCR, qRT-PCR or whatever (excluding those corresponding to the RT). if you get a band, you have DNA in your samples.
I agree with Ajay. Treat your samples with DNAse and do a -RT PCR control.
RNA isolation should always be accompanied with a DNAse treatment. Read your samples on a NanoDrop at 260/280, which should give you an indication if the sample has any DNA in it. However these machines do have limits for sensitivity. If you find that your sample appears to have a reading that shows DNA contamination and/or low RNA yield, you can use phenol/chloroform extraction to clean up the sample and concentrate your product.
http://www.mobio.com/blog/2012/08/03/high-quality-rna-in-accurate-results-out-how-to-qc-your-rna/
The ratio of A260/A280 is an indicator of the DNA or protein contamination of RNA samples. However, it is always better to run -RT control. if you find any band, it comes from DNA.
You can also choose carefully a set of primers that span 2 exons of your gene of interrest. If your RNA was contaminated with DNA during cDNA synthesis, you will have in your gel 2 bands, one will be your expected product (coming from RNA) and a bigger band (coming from DNA, if contaminated).
The problem is, in Bacteria such as E.coli or K. Pneumonia, there is no extron for checking by the PCR. Therefore, it is good practice to have negative sample ( without Reverse Transcriptase) and use it to evaluate the intensitivity of the bands with appropriate thermal cycle between negative and positive samples.
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