How can you figure out the copy number of genes in a cancer cell line without using Southern blotting and whole genome sequencing?
Does anybody try any alternative ways? Any advice is greatly appreciated. Thx.
Look around your University for someone who has a droplet digital PCR system from Bio-Rad. Use info in their application guide to to develop an EvaGreen assay based on primers for the target and two or more reference genes (RPP30 is one and I use SRY because I work with males). Better yet, work out something me to do the work here at Penn State.
Look around your University for someone who has a droplet digital PCR system from Bio-Rad. Use info in their application guide to to develop an EvaGreen assay based on primers for the target and two or more reference genes (RPP30 is one and I use SRY because I work with males). Better yet, work out something me to do the work here at Penn State.
Which cell line are you using? You may also do FISH using a gene/locus-specific probe, which will give you at least an indication of how many copies of the genomic region are present.
Multiplex ligation-dependent probe amplification might be a choice. It's certainly the best way to do copy number analysis but it's Worth to set up only for routine analyses. Otherwise classic PCR might do but you should select several genes with known copy number for comparison and perhaps more than just one amplicon for your region of interest. You also should carefully check primer efficiency and integrate the measured efficiency in your calculation (for example with qGene).
http://en.wikipedia.org/wiki/Multiplex_ligation-dependent_probe_amplification
Thanks all for your insight. I've been told by Dr. McEachron to use Agilent CGH, below.
"You can use array CGH. This is kind of like a microarray designed against
specific regions of the genome. You look at the signal generated from a
universal pooled reference DNA versus the sample of interest. If the
signal for your sample is greater than the reference that means there is a
copy number gain, if the value for the sample is less than the reference,
that means that you have a loss." Any other ways? Thx.
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