It's about an X-linked recessive disorder where there are two affected male siblings and one unaffected female sibling in the family. Upon gene screening by PCR amplification, the whole gene associated with the disease was found to be deleted in both the affected siblings. We would like to validate the copy number variation of the gene in the affected and carrier individuals of the family using SYBR Green Real-time PCR assay. Would it be appropriate to apply relative quantification or absolute quantification to determine the copy number status? Please help me to design the experiment as I am a beginner in real-time PCR analysis
I do copy number analysis in transformed somatic embryos of Pinus pinaster for two years. I do it like described in this two publication by D’haene et al.
Accurate and objective copy number profiling using real-time quantitative PCR. 2010, Methods, Volume 50, Issue 4, April 2010, Pages 262–270.
Corrigendum to “Accurate and objective copy number profiling using real-time quantitative PCR” [Methods 50 (2010) 262–270] Methods, Volume 53, Issue 3, March 2011, Page 326.
For the calculation I use qBase, a very nice software for all qPCR experiments.
If you have more questions we can discuss it later more detailed.
Have a nice weekend.
Kind regards.
http://www.sciencedirect.com/science/article/pii/S104620230900262X
Our bioinformatics unit will do it for a fee. They charge ~$50/h, but since they know what they are doing it's not going to be too many hours.
Usually, just delta-deltaCT for relative quantification of your target region vs. a control region (and ignoring any possible efficency differences) should work, like they did in my link (they used a detection probe instead of CYBR Green, but that does not make any difference).
Others have used standard curves e.g. http://www.ncbi.nlm.nih.gov/pubmed/14974082 . The advantage of that is not so much an "absolute" quantification (it's only absolute if you really know the concentration / copy number of your control standard curve) but the possibility to add amplification efficiency into the equation. But again, usually you don't need that. If you have the mother, you have an obligate carrier (unless there is germline mosaicism) that serves just fine as positive control for 50% dosage of your gene vs. control.
Primer design is important. They need to be 100% specific, so run your target sequences through repeat-masker, thats the best way to avoid repetitive junk (better than the rep masking in Primer3!). Product size for SybrGreen qPCR should be around 100-200 bp, I like to keep them on the short side, TM of primers around 60-63°, optimally all primer pairs in one experiment (target and control region) differ by no more than 1°C. You can aditionally check Primer specificity in NCBI Primer-Blast (or let that just pick the primers, in advanced settings, you can set everything you can in Primer3).
It's not really hard. Most critical part in my hands has been Primer specificity, DNA quality and very precise pipetting.
And finally, you could take a look at mrc-holland www.mlpa.com and see if they have an MLPA-kit for your gene. That would make it even easier and cost is not that high.
http://www.ncbi.nlm.nih.gov/pubmed/12634865
http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker
http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome
I do not understand why you need to assess copy number by other means when the deleted gene = 0 copies.
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