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IS-RT-PCR assay detection of MT-MMP in a human breast cancer cell line

Authors
Publisher
Academic Press
Publication Date
Keywords
  • Beta Actin
  • Metalloproteinase
  • Article
  • Breast Carcinoma
  • Controlled Study
  • Gene Amplification
  • Housekeeping Gene
  • Human
  • Human Cell
  • In Situ Hybridization
  • Northern Blotting
  • Reverse Transcription Polymerase Chain Reaction
  • Breast Neoplasms
  • Concanavalin A
  • Dna Primers
  • Female
  • Gene Expression Regulation
  • Neoplastic
  • Histocytochemistry
  • Humans
  • In Situ Hybridization
  • Matrix Metalloproteinases
  • Membrane-Associated
  • Metalloendopeptidases
  • Polymerase Chain Reaction
  • Rna-Directed Dna Polymerase
  • Staining And Labeling
  • Tumor Cells
  • Cultured
Disciplines
  • Biology

Abstract

The in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) is a method that allows the direct localisation of gene expression. The method utilises the dual buffer mediated activity of the enzyme rTth DNA polymerase enabling both reverse transcription and DNA amplification. Labelled nucleoside triphosphates allow the site of expression to be labelled, rather than the PCR primers themselves, giving a more accurate localisation of transcript expression and decreased background than standard in situ hybridisation (ISH) assays. The MDA-MB-231 human breast carcinoma (HBC) cell line was assayed via the IS-RT-PCR technique, using primers encoding MT-MMP (membrane-type matrix metalloproteinase) and human β-actin. Our results clearly indicate baseline expression of MT-MMP in the relatively invasive MDA-MB-231 cell line at a signal intensity similar to the housekeeping gene β-actin, and results following induction with Concanavalin A (Con A) are consistent with our previous results obtained via Northern blotting.

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