Quantitation of CD39 gene expression in pancreatic tissue by real-time polymerase chain reaction

Within the past decade, the field of gene expression analysis has constantly evolved, with numerous technologies being available for RNA quantification, including differential display, serial analysis of gene expression (SAGE), quantitative real-time (qRT) polymerase chain reaction (PCR), and microarrays. Although every technique has its specific application, the high levels of accuracy, reproducibility, sensitivity, and specificity have established qRT-PCR as a standard method for detection and quantification of gene expression. In this chapter, all steps of the qRT-PCR procedure, including purification of total RNA from animal tissues, reverse transcription to complementary DNA (cDNA), and quantification of relative gene expression are discussed. We chose qRT-PCR analysis of CD39 in pancreatic tissue as an example that is applicable to any gene of interest. CD39/ecto-nucleoside triphosphate diphosphohydrolase-type-1 (ENTPD1) is the dominant vascular ecto-nucleotidase that hydrolyzes extracellular nucleotides to integrate purinergic signaling responses. It has recently been associated with tumor growth and proliferation in melanoma cells and linked to pancreatic cancer progression.