I am trying to estimate copy number of a gene (it is multiple copy gene in tandem repeats). But I don't have any sample with known copy number. I am using taqman probes for this study.Is there any other method to calculate copy number in at least one sample ,so that I can use the same as reference for real time quantication?
Not any simple solution I can think of. Likely, you'l need to clone the gene of interest, and use that clone for the calibration. Another possibility is to use a specific HPLC column for separating genes in a tandem upon their sequence, isolate one, relate its MW to obtained signal, and use it as a reference.
Which organism are you analyzing? Knowing this will make it easier to find a solution for your problem.
If you are investigating human, why don't you just take a normal tissue or a cell line, which has been shown to harbor only two copies of your region of interest?
A reference is an absolute must. You can think of using some other single copy gene in the human genome as a reference. By that way you can calculate no. of copies of single copy gene per pg or ng of genomic DNA. Thus for a single copy gene you would expect 2 copies per ~3.5 pg of diploid human genomic DNA. You could then create a calibration curve. Using a reference will also help you make sure the quality of genomic DNA, for example degradation etc.
Thanks every body for your answers.Yes, I am trying construct a standard curve after cloning my target and reference gene in separate plasmids.Based on which I think I have to estimate my target gene copy number.I just wanted to know whether I can use any alternate method like Fiber FISH to estimate the length of the cluster ,hence copy number of my target gene .Because I want to cross check the copy number calculated by standard curve (using taqman assay) with any other method atleast in a couple of samples.My sample size is around 800.
I think you are referring to an absolute copy number quantification method. the best method available now is a digital/droplet PCR. in this method (http://www.youtube.com/watch?v=9TJy_uegmd4).
Digital PCR is a new approach to nucleic acid detection and quantification. It offers a different method for absolute quantification and rare allele detection relative to conventional real-time quantitative PCR. Digital PCR works by partitioning a sample into many individual real-time PCR reactions; some portion of these reactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction of negative reactions is used to generate an absolute count of the number of target molecules in the sample, without reference to standards or endogenous controls.
sensitivity of this method is so high that it outperforms many other methods including Taqman.
Good Luck !!!
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