Hallo Emma,

I normally check for gDNA conatmination by a PCR with primers, which give a different amplification product, when genomic DNA is present. If there is an intron which is spliced out in all the cDNA s you sequenced, you could design primers in that particular region.

Best regards,

Johanna

Hi Emma,

You can use Negative RT as a control.  So, basically, at the time of preparing the cDNA, you can make Negative RT COntrol = RNA (DNase treated) + dNTPS + Random Hexamers/Oligo dT + RT Buffer + Water WITHOUT RT ENzyme. Do the cDNA prep by whatever method you usually do.

Now, use both +RT and -RT samples and now do PCR with the primers for Dystrophin. If you don't have any gDNA contamination, your -RT will be clean and will not give any amplicon. +RT will obviously/should work.

If your -RT control shows any amplicon, this suggests that your DNase treatment of the RNA is not good enough and you need to treat it again.

Hope this helps. Good Luck!

Utkarsh

You should used DNase treatment before cDNA synthesis to remove gDNA and a PCR with a housekeeping  primers which give a suitable amplicon, but if you mean that you did cDNA synthesis you can have a PCR too, if your desired gene is intronic-exonic kind, Ok the amplicon size is different for gDNA and cDNA. Introns dont exist in cDNA so you observe a shorter band rather than gDNA.

Presumably, you don't want to start from the beginning, Dnasing your RNA again (?) or making -RT reactions and going through the whole process again.  But if you still have the original RNA, you can just run your PCR reaction on that to see if you get these "intron-retained" products.  You need to calculate the equivalent amount of RNA that went into the positive cDNA reaction.  If you add that amount to a PCR (and you should also try 2x, 4x, 6x that amount--or perform multiple 1x reactions), and you don't get the same "intron-retained" product, you can present that to the reviewers as evidence that the "intron-retained" product is indeed coming from an RNA that is present in the cell line and not genomic DNA contamination. 

When designing a cDNA specific PCR you should choose your primers on different exons, preferably on exons wit the largest intron in between.

If no introns are present in your gene you can do the things as has been mentioned before by others, However in my experience DNAse treatment does not solve all of the background signal, small DNA fragments can still interact with your PCR, these small fragments can act as primers also and give still PCR products from the gDNA still present in little amounts..

Some responders here don't seem to have read the question carefully.  What Emma has is a cell line which seems to have poor splicing of the dystrophin gene--so the idea is that an abnormal proportion of the dystrophin primary transcripts are not fully spliced.  And she is looking for a way to confirm or test that there is a splicing deficiency. The PCR products from a cDNA transcript of an unspliced  primary transcript RNA  will necessarily correspond exactly to the genomic PCR product of those primers.  The question is, how does she show that any "unspliced " PCR products are derived from an RNA intermediate and not from genomic DNA.  

Having said that, it is unclear from Emma's description whether it seems that all of the dystrophin introns are being spliced out poorly or only whether specific introns are.  If the latter, then another approach might be to design primers that span the location of a well-spliced intron and a poorly spliced one.  The presence of the correct splice junction in the resulting product would show that it is RNA derived and therefore that the unspliced portion is also RNA-derived. One could then compare the products from that cell line with another sample that does not have the splicing deficiency to make a quantitative estimate of the effect.

Use any house keeping gene and do PCR with your RNA(The way you use DNA). If there is DNA contamination you should get amplification.

I would one negative control called minus RT where it is basically do cDNA synthesis without the RT enzyme and do qPCR. If you have amplification it means that this is because possible genomic DNA contamination. 

To be sure that there is no genomic DNA, I would recommend use DNAseI treatment.