A quantitative assay for IGF-I and IGF binding protein mRNAs: expression in malignant melanoma cells.
The quantification of messenger RNA is central in studies of gene expression. We describe a quantitative assay for specific mRNAs (QASM) that measures mRNAs for insulin-like growth factor-I, IGF binding proteins (IGFBPs) -2, -3, -4, and -5, and beta-actin. The assay utilizes reverse transcription and polymerase chain reaction, followed by an ELISA based DNA assay technique. The use of internal (competitive) quantification standards gave poorly linear results, while external standards gave linear and reproducible results. QASM results correlated with IGFBP protein concentrations in conditioned medium and with mRNA levels determined by Northern blotting. QASM was used to study IGFBP expression in human malignant melanoma cells. Messenger RNA for IGFBP-2, -3, and -5 were present, while IGF-I and IGFBP-4 mRNAs were not detected. IGFBP-2 and -3 expression was increased in a dose dependent manner by treatment with IGF-I. Protein concentrations in conditioned media paralleled mRNA levels. QASM is a sensitive, specific, and reproducible approach to determining mRNA levels.
Olney
R C
RC
Department of Pediatrics, Stanford University Medical Center, CA 94305, USA.
Anhalt
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Neely
E K
EK
Wilson
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DM
D000199:Actins / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics*; D017077:Culture Media, Conditioned; D015870:Gene Expression; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2; D018975:Insulin-Like Growth Factor Binding Protein 5; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D008545:Melanoma / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D014407:Tumor Cells, Cultured
D000199:Actins; D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D018971:Insulin-Like Growth Factor Binding Protein 2; D018975:Insulin-Like Growth Factor Binding Protein 5; D018969:Insulin-Like Growth Factor Binding Proteins; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D012194:RNA-Directed DNA Polymerase