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.
- PCR of Gene Rearrangements for the Detection of Minimal Residual Disease in Childhood ALL
- Hormones as Biomarkers: Practical Guide to Utilizing Luminex Technologies for Biomarker Research
- Epigenetics in Ovarian Cancer
- Bcl-2 Family Immunohistochemistry
- Mutational Analysis of the Wilms' Tumor (WTI) Gene
- Detecting and Modulating the NF-kB Activity in Human Immune Cells: Generation of Human Cell Lines with Altered Levels of NF-B
- Cloning Differentially Expressed Genes Using Rapid Subtraction Hybridization (RaSH)
- Detection of MicroRNAs in Cultured Cells and Paraffin-Embedded Tissue Specimens by In Situ Hybridization
- Measurement of Multiple Drug Resistance Transporter Activity in Putative Cancer Stem/Progenitor Cells
- 软琼脂克隆形成实验