Studying copy number variations using a nanofluidic platform

Qin, Jian; Jones, Robert C.; Ramakrishnan, Ramesh

Copy number analysis using the digital array on the BioMark system Each panel of a digital array contains a total of 4.59 ml (6 nl  765 chambers) PCR reaction mix. However, 10 ml reaction mixes were normally prepared for each panel, containing 1  TaqMan gene expression master mix (Applied Biosystems, Foster City, CA), 1  RNase P-VIC TaqMan assay, 1  TaqMan assay (900 nM primers and 200 nM probe) for the target gene, 1  sample loading reagent (Fluidigm, South San Francisco, CA) and DNA with about 1100-1300 copies of the RNase P gene. The reaction mix was uniformly partitioned into the 765 reaction chambers of each panel and the digital array was thermocycled on the BioMark system (http:// www.fluidigm.com/products/biomark-main.html). Thermocycling conditions included a 958C, 10 min hot start followed by 40 cycles of two-step PCR: 15 s at 958C for denaturing and 1 min at 608C for annealing and extension. Molecules of the two genes were independently amplified. FAM and VIC signals of all chambers were recorded at the end of each PCR cycle. After the reaction was completed, Digital PCR Analysis software (Fluidigm, South San Francisco, CA) was used to process the data and count the numbers of both FAM-positive chambers (target gene) and VIC-positive chambers (RNase P) in each panel.