Advanced qRT-PCR technology allows detection of the cholecystokinin 1 receptor (CCK1R) expression in human pancreas

• Published in: Pancreas Vol. 31; no. 4; p. 325
• Main Authors: Galindo, Jose, Jones, Neil, Powell, Garth L, Hollingsworth, Simon J, Shankley, Nigel
• Format: Journal Article
• Language: English
• Published: United States 01.11.2005
• Subjects: DNA, Complementary - genetics; Humans; Pancreas - metabolism; Receptor, Cholecystokinin A - genetics; Reverse Transcriptase Polymerase Chain Reaction - methods
• ISSN: 1536-4828
• DOI: 10.1097/01.mpa.0000181487.50269.dc

To help clarify the controversy over the detection of expression of the cholecystokinin 1 receptor (CCK1R; CCKAR) in human pancreas. Applied qRT-PCR to detect CCK1R expression using the SYBR green/Smart Cycler II and the QZyme oligonucleotide/ABI PRISM 7500 systems to detect CCK1R expressed message in highly purified cDNAs from human pancreas and other tissues. Samples of normal pancreas were obtained at operation (pancreaticoduodenectomy; Whipple's procedure) and used to ascertain the expression of CCK1R in human tissue and investigate donor individual variability in expression levels by semi-quantitative RT-PCR and scanning densitometry. We present molecular evidence obtained with advanced qRT-PCR technology that clearly establishes CCK1R expression in human pancreas. Amplification variation in individual human samples is documented here. By targeting different stretches of the sequence with several primer pairs, it was observed that SYBR green qRT-PCR failed to amplify efficiently over GGA-and GAA-rich nucleotide triplet regions, leading to false negative results. The QZyme system quantified the expression with the following distribution: stomach > small intestine approximately colon > brain approximately kidney > pancreas. CCK1R expression levels varied from undetectable, to high levels of expression, in individual samples collected from surgical specimens. CCK1R message can be conclusively detected and quantified in human pancreas cDNA by targeting the appropriate nucleotide sequence regions of this gene.