title: Development of a Quantitative PCR Assay for Detection of Human Insulin-Like Growth Factor Receptor and Insulin Receptor Isoforms.
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.
authors: 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.
authors: Clare A
authors: CA
authors: Flannery
authors: 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.
authors: Anne M
authors: AM
authors: Rowzee
authors: 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.
authors: Gina H
authors: GH
authors: Choe
authors: 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.
authors: Farrah L
authors: FL
authors: Saleh
authors: 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.
authors: Caitlin C
authors: CC
authors: Radford
authors: 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.
authors: Hugh S
authors: HS
authors: Taylor
authors: 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.
authors: Teresa L
authors: TL
authors: Wood
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000235:genetics*; D011972:Receptor, Insulin / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D020033:Protein Isoforms; D012333:RNA, Messenger; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin