Real-time qPCR was conducted with the BioMark TM Dynamic Array Microfluidics System (Fluidigm Corporation, South San Francisco, CA) using RNA isolated from frozen pancreatic tissues. First, total RNA was extracted from frozen slide sections of pancreatic tissue classified as normal (NAT, normal adjacent to tumor), adenocarcinoma, benign adenoma, or endocrine tumor using the ZR RNA MicroPrep kit (Zymo Research, Orange, CA). RNA purity and concentration were determined spectrophotometrically. RNA quality was assessed on an Agilent 2100 Bioanalyzer using the RNA 6000 Nano LabChipH (Agilent Technologies, Santa Clara, CA). Single stranded cDNA was generated from total pancreas RNA using the SuperScriptH III First-Strand Synthesis SuperMix (Invitrogen). cDNA samples were pre-amplified using TaqMan Pre-Amp Master Mix (Invitrogen), according to the manufacturer's instructions. Reactions were cycled with the recommended program for 14 cycles and then diluted 1:5 with TE buffer. Preamplified cDNA was either utilized immediately or stored at 220uC until processed. Samples and TaqMan Gene Expression assays (Applied Biosystems, Carlsbad, CA) were load onto a 96.96 dynamic array (Fluidigm) according to the manufacturer's instructions. The prepared array was loaded on the BioMark TM Real-Time PCR System (Fluidigm) for thermal cycling (10 min at 95uC followed by 40 cycles of 95uC for 15 sec and 1 min at 60uC). Upon completion of the qPCR, Fluidigm Real-Time PCR Analysis software was used to generate threshold cycle (Ct) values. A Ct cutoff value of 24, below which the samples were considered to be positive and above which the samples were considered to be negative for transcript expression, was empirically selected.