The CLIP-CHIP oligonucleotide microarray allows the analysis of mRNA transcript levels in a tissue sample for all proteases, nonproteolytic homologs, and protease inhibitors of the human and mouse genome. In the protocol presented in this unit, total RNA is extracted from a tissue, and the resulting mRNA is reverse transcribed into cDNA and dsDNA and then amplified in an in vitro transcription reaction. The amplified antisense RNA is labeled with a fluorescent dye and hybridized to the CLIP-CHIP, which contains unique oligonucleotides that are specifically designed for the protease, nonproteolytic homologs, protease inhibitors, and control samples. After hybridization, the fluorescence intensity of each spot is measured, thus identifying mRNA transcripts that are expressed and allowing basic quantification of expressed transcripts.
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http://dx.doi.org/10.1002/0471140864.ps2119s56 | DOI Listing |
Biochim Biophys Acta Mol Cell Res
November 2017
Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia, Canada; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada. Electronic address:
The protease degradome is defined as the complete repertoire of proteases and inhibitors, and their nonfunctional homologs present in a cell, tissue or organism at any given time. We review the tissue distribution of virtually the entire degradome in 23 different human tissues and 6 ovarian cancer cell lines. To do so, we developed the CLIP-CHIP™, a custom microarray based on a 70-mer oligonucleotide platform, to specifically profile the transcripts of the entire repertoire of 473 active human proteases, 156 protease inhibitors and 92 non-proteolytically active homologs known at the design date using one specific 70-mer oligonucleotide per transcript.
View Article and Find Full Text PDFJ Biol Chem
September 2011
Centre for Blood Research, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Dynamic reciprocal interactions between a tumor and its microenvironment impact both the establishment and progression of metastases. These interactions are mediated, in part, through proteolytic sculpting of the microenvironment, particularly by the matrix metalloproteinases, with both tumors and stroma contributing to the proteolytic milieu. Because bone is one of the predominant sites of breast cancer metastases, we used a co-culture system in which a subpopulation of the highly invasive human breast cancer cell line MDA-MB-231, with increased propensity to metastasize to bone, was overlaid onto a monolayer of differentiated osteoblast MC3T3-E1 cells in a mineralized osteoid matrix.
View Article and Find Full Text PDFMethods Mol Biol
April 2010
Department of Oral Biological and Medical Sciences, Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada.
The degradome microarray - CLIP-CHIP - is a dedicated and focused array that allows the analysis of all proteases, non-proteolytic homologs, and protease inhibitor gene transcripts in the human and murine genomes at the mRNA transcript level.Based on unique 70-mer oligonucleotides, designed to match parts of the sequence of known or predicted protease and inhibitor mRNAs in both species and printed on a glass-matrix surface, the CLIP-CHIP microarray can be used to analyze differentially expressed protease and inhibitor gene products and give expression profiles for any analyzed sample.
View Article and Find Full Text PDFCurr Protoc Protein Sci
April 2009
University of British Columbia, Vancouver, British Columbia, Canada.
The CLIP-CHIP oligonucleotide microarray allows the analysis of mRNA transcript levels in a tissue sample for all proteases, nonproteolytic homologs, and protease inhibitors of the human and mouse genome. In the protocol presented in this unit, total RNA is extracted from a tissue, and the resulting mRNA is reverse transcribed into cDNA and dsDNA and then amplified in an in vitro transcription reaction. The amplified antisense RNA is labeled with a fluorescent dye and hybridized to the CLIP-CHIP, which contains unique oligonucleotides that are specifically designed for the protease, nonproteolytic homologs, protease inhibitors, and control samples.
View Article and Find Full Text PDFCurr Protoc Protein Sci
August 2007
University of British Columbia, Vancouver, British Columbia, Canada.
The CLIP-CHIP oligonucleotide microarray allows the analysis of mRNA transcript levels in a tissue sample for all proteases, nonproteolytic homologs, and protease inhibitors of the human and mouse genome. In the protocol presented in this unit, total RNA is extracted from a tissue, and the resulting mRNA is reverse transcribed into cDNA and dsDNA and then amplified in an in vitro transcription reaction. The amplified antisense RNA is labeled with a fluorescent dye and hybridized to the CLIP-CHIP, which contains unique oligonucleotides that are specifically designed for the protease, nonproteolytic homologs, protease inhibitors, and control samples.
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