title: Wafergen Biosystems SmartChip Human Oncology Panel v2
status: Public on Jul 01 2012
contact.city: New York
contact.first: Zeynep
contact.middle: H.
contact.last: Gumus
contact.email: zeg2002@med.cornell.edu
contact.state: NY
contact.address: 1300 York Ave
contact.department: Physiology and Biophysics
contact.country: USA
contact.institute: Cornell University
organism: Homo sapiens
description: SYBR Green qPCR assay
manufacture_protocol: 1239 unique primer pairs in quadruplicate were printed on nano volume SmartChip for a total of 5184 wells. There are 11 endogenous control and 4 exogenous control primers. Total RNA was analyzed using the SmartChip Real-Time PCR System (WaferGen Biosystems, Fremont, CA). Briefly, cDNA was prepared using 1 ug of total RNA per sample per manufacturer’s recommendation. A PCR cocktail containing SYBR Green I dye and the equivalent of 1000 ng of starting RNA for each sample was loaded onto the SmartChip Human Oncology V2 Panel (containing 1,239 unique real-time PCR reactions in quadruplicate for a total of 5,184 reactions/sample.) The volume per reaction was 100 nL with an equivalent of 96 pg of RNA loaded per reaction. Forty cycles of real-time PCR were performed on the SmartChip Cycler collecting both raw Ct and Tm of each gene and sample for data analysis.  A data quality screen on amplification, Tm curves, and Ct and Tm variability was performed to remove any outlier data before delta-delta Ct calculations were used to determine fold change in expression.