Format

Send to

Choose Destination
Endocrinology. 2016 Apr;157(4):1702-8. doi: 10.1210/en.2015-1698. Epub 2016 Feb 10.

Development of a Quantitative PCR Assay for Detection of Human Insulin-Like Growth Factor Receptor and Insulin Receptor Isoforms.

Author information

1
Department of Obstetrics and Gynecology and Reproductive Sciences (C.A.F., G.H.C., F.L.S., C.C.R., H.S.T.), Yale School of Medicine, New Haven, Connecticut 06520; and Department of Pharmacology, Physiology, and Neuroscience and Cancer Center (A.M.R., T.L.W.), New Jersey Medical School, Rutgers University, Newark, New Jersey 07103.

Abstract

The biological activity of insulin and the insulin-like growth factor (IGF) ligands, IGF-I and IGF-II, is based in part on the relative abundance and distribution of their target receptors: the insulin receptor (IR) splice variants A (IR-A) and B (IR-B) and IGF 1 receptor (IGF-1R). However, the relative quantity of all three receptors in human tissues has never been measured together on the same scale. Due to the high homology between insulin receptor (IR)-A and IR-B proteins and lack of antibodies that discern the two IR splice variants, their mRNA sequence is the most reliable means of distinguishing between the receptors. Hence, highly specific primers for IR-A, IR-B, and IGF-1R mRNA were designed to accurately detect all three receptors by quantitative RT-PCR and enable direct quantification of relative receptor expression levels. A standard concentration curve of cDNA from each receptor was performed. Assay specificity was tested using competition assays and postamplification analysis by gel electrophoresis and cloning. Forward and reverse primer concentrations were optimized to ensure equal efficiencies across primer pairs. This assay enables a specific molecular signature of IGF/insulin signaling receptors to be assayed in different tissues, cell types, or cancers.

PMID:
26862994
PMCID:
PMC4816738
DOI:
10.1210/en.2015-1698
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Silverchair Information Systems Icon for PubMed Central
Loading ...
Support Center