A major aim in using real-time PCR is to allow accurate quantification of gene modifications in DNA extracted from relatively few cells. For this purpose, the number of cells present in a given sample can be estimated using real-time PCR quantification of the copy number of a 'reference' gene, each cell having two allelic copies of this gene. In principle, any gene is suitable but examination of deletions, amplifications and rearrangements requires this 'reference' gene not to be involved in any of these processes. The genetic abnormality is then quantified relative to this reference.
It is necessary to establish the validity of the relationship between the amount of DNA in the sample and the 'reference' Ct reading. The relationship between the number of cells in a sample and the DNA concentration of the sample also needs to be linear, indicating a similar efficiency of the DNA extraction method irrespective of the initial number of cells in the sample.
Archived material such as paraffin embedded tissue sections can be used. DNA can be extracted from such material using a simple proteinase K and phenol extraction methodology, with no need for de-waxing, and used in a SYBR® Green I real-time DNA quantification assay.
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