Quantitative PCR Protocol

Quantitative PCR Protocol

Don Liu, Cecilia Schmidt, Tim Billings, and Muriel T. Davisson, The Jackson Laboratory

Genotyping Ts65Dn mice is based on doing simultaneous quantitative PCR amplification of a gene or genes in the Ts65Dn chromosome and a control gene on another chromosome (in this case Apob) and comparing the average change (delta) in threshold cycle (CT) between the Ts65Dn genes and the control gene. Doing a multiplexed reaction with an internal control avoids the need for determining DNA concentration and permits a relatively large range of variation in concentration of template DNA.

Although trisomic females vary in transmission of the trisomy the overall incidence of trisomy among progeny is only 19-25%. To streamline the typing process further the animals may be visually phenotyped before they are genotyped. The phenotype is variable and subtle but to the trained eye the trisomic mice can be identified. Ts65Dn mice are usually smaller than their control littermates at weaning. When one lifts the cage cover slowly, the trisomic mice often raise their heads and bring their ears to the side. When picked up by the tail some trisomic mice make a high-pitched continuous squeak. Ts65Dn mice often display stereotypic behavior, repeatedly jumping up and down by the side of the cage.  When a pencil is placed vertically in front of the mouse, controls will run around it but Ts65Dn mice sit back on their haunches and "chatter". Phenotyping the mice is not 100% accurate nor should it be relied upon to classify mice, but it does help eliminate control animals that are not needed and reduce the number of mice that must be genotyped. The combination of visual phenotyping followed by qPCR genotyping should make identifying Ts65Dn mice easier than chromosomal typing for most researchers.

Full protocol here

http://www.jax.org/cyto/quanpcr.html