3 replies to this topic

[dna_nerd]

hello all

is it possible to determine copy number variation of a given gene using genomic dna samples using qPCR?

I would like to compare a couple genes for a group of patients, can I use SYBR or TaqMan?

thanks

[Trof]

Yes it's possible.
You just need a reference gene, one you know certainly is single-copy, to normalize to. You can use either method, TaqMan offers higher specificity, but is more expensive.

[dna_nerd]

Trof, on 14 October 2010 - 05:56 AM, said:

Yes it's possible.
You just need a reference gene, one you know certainly is single-copy, to normalize to. You can use either method, TaqMan offers higher specificity, but is more expensive.

Trof -

Thanks for the reply, I'm going to get some more info out of you if thats alright.

Since I am looking at genomic DNA I assume my reference sample would need to be genomic as well?  Since I am looking at copy number variation in canine oncology patients possibly a disease free patient would work.

Once I have a reference sample would  the experiment be performed as an absolute or relative quantification (do I need a standard curve of the reference gene or would it act as my "untreated sample" and compare them both to a housekeeping gene)?

Thanks

[Trof]

You're either going to compare patient and control - "healthy" samples together (both gDNA and preferentially both isolated same way), hence you're doing relative quantification and you can choose from several methods (site comparing these, or search the forum) but basically it's better to use Pfaffl method and standard curve made from serial dilutions. Then you need reference - "housekeeping" single-copy gene and control sample/samples.

Or you can make standards of the known copy-number (usualy plasmids) to run with your reaction and do absolute quantification. But you still need to normalise it to a reference gene, because your input concentration may vary a little (and you need the standards for this gene too). This is usually done by taking those two calculated copy numbers and divide them to give a ratio of your target/reference gene.
You don't need control samples to do this, if you know the copy number in normal population (say 2).