4 replies to this topic

[rajah]

Hello,

I´d like to ask about different ways to check genomic contamination from cDNA samples.

Of course, -RT samples can be done before qPCR or in qPCR and run in gel, but both, especially latter get quite costly because of the excess use of master mixes (if there are lots of samples). I´ve also heard that contamination could be check by traditional PCR and using specific primers for example for Hdac7a (histone deacetylase 7a). Anyone heard of this?

Thanks for help!

[leelee]

I would just do the -RT controls. Yes they might be expensive, but you data means NOTHING without them. A positive PCR result is meaningless without the right controls to validate it.

Even running a seperate standard PCR to check for contamination wouldn't be good enough in my opinion. Your primers are different, your cycle conditions are different and so on, so its not a good control for your qPCR at all.

[Trof]

You can get rid of any possible gDNA contamination if you design intron-spanning primers and/(or) use a DNase treatment.  We use both and only run a RT- once in a while to check it really works, and it's always negative.

[rajah]

Trof, on Jun 7 2010, 02:41 PM, said:

You can get rid of any possible gDNA contamination if you design intron-spanning primers and/(or) use a DNase treatment.  We use both and only run a RT- once in a while to check it really works, and it's always negative.

At the moment, it is impossible for me to design intron-spanning primers. Therefore, I ofcourse use a DNAse treatment.

But what I´m talking about is primers designed to test for gDNA contamination. Intron-spanning, so that it gives a shorter product from cDNA and a longer one from possible gDNA, do regular PCR and then check the products on gel.

Make any sense?  

[Trof]

rajah, on Jun 7 2010, 04:54 PM, said:

At the moment, it is impossible for me to design intron-spanning primers. Therefore, I ofcourse use a DNAse treatment.

But what I´m talking about is primers designed to test for gDNA contamination. Intron-spanning, so that it gives a shorter product from cDNA and a longer one from possible gDNA, do regular PCR and then check the products on gel.

Make any sense?  

If you want to see gDNA contamination you can design primers for virtualy any gene. But it would make sense to use the gene of interest or your housekeeping gene, but that really doesn't matter that much AFAIK. Intron spanning primers (each primers in different exon) can be used to see both your product and genomic contamination in the same run while using SYBR (by melting curve analysis). Otherwise you can just make primers for gDNA only, i.e. in introns, that would not amplify any cDNA, this would be more sensitive for gDNA than intron spanning assay. This, however, has a few BUTs..

You're not testing in the same conditions, I would be affraid that if you simply try to amplify gDNA from your RNA sample, you could find that there is always some traces of gDNA left, specialy if you use a sensitive method, but that doesn't mean it's amplifying in your qPCR reaction too. That's why is RT- done, because the RT mix has some inhibition potencial and you use exactly the same conditions to run it.  What you really want to know is, if the gDNA is somehow affecting your qPCR results and that can only be determined by running a RT- sample.
But of course, if you test your validated (with estimated detection limit) intron gDNA assay on your RNA on real-time PCR (more sensitive than classic PCR and gel) and it comes negative, then you're sure there's no gDNA contamination. But what if it comes very slightly positive, how can you tell if it affects your qPCR results?


(Just to clarify, there are two types of intron-spanning assays, one has each primer on different exons with a shorter intron between them, that could detect both cDNA and gDNA differing in product legth, and intron-spanning primer (or probe) that lies on the junction of two exons in cDNA, these don't amplify gDNA at all even if present)