Dear Azaz,

You tackle several problems here.

About the question of gDNA contamination - there are at least three ways to find out if you have gDNA in your sample.

1) You run a gel - as you did. No prominent band above the 28s peak, but something is there. Could be gDNA.

2) You look at the electropherogram of your Bioanalyzer measurement.

You talked about integrity of your RNA but didn´t mention if you have measured it. Just in general - the integrity number (=RIN value) tells you how intact your RNA is and is only a measure to compare different samples. The idea behind it is, that you should not compare samples with strikingly different RIN values as this can give a false impression about the amount of transcript for your genes of interest.

If you check the electropherogram of your samples you can see it if considerable amounts of DNA are present - there will be a large peak before the 28s-RNA peak.

3) You run a PCR with your RNA samples, preferably with a highly abundant/transcribed gene. If you get an amplification you certainly had gDNA in this one. On the other hand, no amplification does not mean there is no gDNA in your samples.

If you have enough material I´d recommend you do a DNAse digest as recommended above. If you feel nifty do the -RT control as Thomas suggested as well. Then run your qPCR again and see what happens.

By the way, in accordance to the MIQE guidelines you will have to proof your samples are free of (considerable amounts) of gDNA if you want your results to get published.

Good luck.

You could consider taking a small amount and carrying out a DNAse digestion on it. Then run similar amounts of DNase treated and untreated samples. If the top band disappears, it would imply it is genomic.

thanks @sandeepa I never thought about it this is another good way to know it.

I would guess that the high molecular weight band is gDNA based on your agarose gel. You could perform DNaseI treatment of the RNA before the RT step (I recommend Ambion TURBO Dnase for this). The easiest way to test for gDNA contamination is to perform an RT- control. This is a mock reverse transcription containing no RT enzyme. Amplification in this reaction is usually indicative of gDNA contamination. An unofficial rule-of-thumb is that you can disregard gDNA contamination (when doing relative quantification) if the gDNA amplifies >5 cycles after the cDNA amplification (i.e. >32 fold less). This of course depends on your experiment. Another strategy you can use to avoid amplifying gDNA is to design your qPCR primers so that they span an exon-exon junction. If you pick a suitably large intron the distance between the primers in gDNA space will be too great for amplification to occur.

We regularly do on column dnase digestion with the qiagen or ambion ra mini colums. It is easy, quick (15 mins) and cheap. I use the RNAse free DNAse from Qiagen. No worries about DNA contamiation then.

To make shure is your rna is contaminated by dna just do simple thing i.e. Put pcr with housekeeping gene using your rna as a template. Run product on agarose gel if you see a band then there is a dna contamination in your rna sample..because rna cant be a template..

Dear Azaz,

You tackle several problems here.

About the question of gDNA contamination - there are at least three ways to find out if you have gDNA in your sample.

1) You run a gel - as you did. No prominent band above the 28s peak, but something is there. Could be gDNA.

2) You look at the electropherogram of your Bioanalyzer measurement.

You talked about integrity of your RNA but didn´t mention if you have measured it. Just in general - the integrity number (=RIN value) tells you how intact your RNA is and is only a measure to compare different samples. The idea behind it is, that you should not compare samples with strikingly different RIN values as this can give a false impression about the amount of transcript for your genes of interest.

If you check the electropherogram of your samples you can see it if considerable amounts of DNA are present - there will be a large peak before the 28s-RNA peak.

3) You run a PCR with your RNA samples, preferably with a highly abundant/transcribed gene. If you get an amplification you certainly had gDNA in this one. On the other hand, no amplification does not mean there is no gDNA in your samples.

If you have enough material I´d recommend you do a DNAse digest as recommended above. If you feel nifty do the -RT control as Thomas suggested as well. Then run your qPCR again and see what happens.

By the way, in accordance to the MIQE guidelines you will have to proof your samples are free of (considerable amounts) of gDNA if you want your results to get published.

Good luck.

2 ways: Kit method: you can also work up with Qiagen kit; and/or Manual method: you work up with phenol extraction from 100 micro to 1000mcrolitres. Take care while collecting aquious phase without white pricipitate and add lesser than equal amount Ethanol for precipitation and Keep Work area seperate for DNA extraction and PCR to escape contomination

Definitely looks like gDNA contamination in your gel (although quite sharp band and not diffuse) Just do DNAse treatment, I use Ambion DNA Free kit.

You can digest it by DNAse for twice. Nomally, it works very well. After this digestion, you can try to do some PCR, or QPCR to check it.

I have found the Roche kit is excellent for eliminating genomic DNA - was having problems with Qiagen kit for my samples. Definitely run an RT control in the absence of RT enzyme for a housekeeping gene preferably and this will confirm/negate any suspicions you have.

The sharp bands located above the 28S band in your gel photo are very likely to be rRNA processing intermediates--the rRNA locus containing 28S, 18S and 5S segments is transcribed in a single primary transcript and then processed.

To assess gDNA contamination you can simply do a genomic qPCR on your RNA or your cDNA. Use primers that only amplify a genomic product. You can make a standard curve if you like with pure genomic DNA.

DNAsing is necessary if your RT-qPCR primers amplify a genomic segment and your gene of interest is not highly expressed. You can determine this by running the -RT reaction as others have suggested. If removing ALL DNA becomes necessary, I would not rely on "on-column" DNAsing methods, since in my hands such products from various companies have been only weakly effective in reducing DNA template amounts.

All the above suggestions are very important and ensure to get rid of DNA from your sample or gel . What we use to practice is to design INTRON Spanning primers. So that if Genomic DNA is present, primer would partially anneal but wont yield the product. Secondly if primer gets annealed perfectly , the size of product with Genomic DNA would be different as compared to the cDNA.

Any RNA extraction method co-purifies DNA to some extent (usually around 1%, but can be up to 10%). It is highly recommend to do a DNase treatment, either on the column or after RNA purification on the RNA. To some extent, DNA co-amplification during RT-qPCR can be avoiding by careful primer design (intron needs to be at least 700 bp when using an intron spanning assay; junction assays work too but are more difficult to design). Unfortunately, this design strategy does not work for at least 15% of the human genes because processed pseudogenes (retropseudogenes, intronless copies of the original gene) reside in the genome. That is why we always recommend to do a DNase treatment. We now use the Heat and Run kit from Arcticzymes when removing DNA from RNA (http://arcticzymes.com/technology/kits/heat-and-run/). Importantly, after DNase, you need to verify that the RNA is devoid of DNA. Just run a qPCR with DNA-specific primers (i.e. that do work on DNA) on RNA. As RNA is no template for PCR, any signal you get is from contaminating DNA.

Hi Azaz,

Your pic surely looks like there is gDNA, and no matter what method you follow for RNA isolation you will still see traces of DNA in it. So, I suggest you to carry out DNase treatment with NEB or Ambion regaent but do not forget to add EDTA as without it your RNA will get degraded. It is known that 10% of the product you end with in your final RNA preparation will be DNA, so go ahead and digest it and get rid of it to ensure clean results with your qPCR.

All the best

Madhavi

You can not determine whether there is trace gDNA contamination by the gel, You can only evalucate the RNA degradation. The best way to test the presence of gDNA is negative RT-PCR . Run a PCR using house keeping gene primers without cDNA reverse trascription. If you can not see any band amplified after 35 cycle, then you can say your RNA is free of gDNA. Remember to run a positive control (with reverse trascription) as well. Hope it helps.

I used Experion DNA Analysis Kits or PCR amplification  to detect DNA contamination. It is much better than nanodrop. It is very hard to check the DNA contamination by gel.

Conventional RNA purification with TRIZOL can always carry DNA dissolved in the aqueous phase. Technically, see that you don't take much from close to interphase which will only leave you with traces of DNA that can be treated using commercially available DNase. Besides do the DNase treatment on column so that you have a better result. Secondly, always check for  A260/280 ratio during quantification to reassure presence RNA alone. Third, do a simple gel electrophoresis (0.7-1.0%) to check for gDNA. Fourth, use a cDNA synthesis kit that has an in-supplied gDNase. Finally, try to stick to primers spanning the exon junctions for your RT-PCR experiment.

When you have this standard steps followed you don't have to worry on DNA contamination in RNA.

Concerning DNA contamination in RNA samples. We described a single multi-purpose PCR-based integrity assay amplifying multiple fragments: doi: https://doi.org/10.1101/168195. It can perfectly be used to check the level of DNA contamination/degradation in RNA samples. Don't hesitate to contact me for feedback.