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.

Download full-text PDF

Source
http://dx.doi.org/10.1002/0471140864.ps2119s56DOI Listing

Publication Analysis

Top Keywords

clip-chip oligonucleotide
8
oligonucleotide microarray
8
protease nonproteolytic
8
human mouse
8
nonproteolytic homologs
8
homologs protease
8
protease inhibitors
8
microarray dedicated
4
dedicated array
4
array analysis
4

Similar Publications

Overview of transcriptomic analysis of all human proteases, non-proteolytic homologs and inhibitors: Organ, tissue and ovarian cancer cell line expression profiling of the human protease degradome by the CLIP-CHIP™ DNA microarray.

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 PDF

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 PDF

Analysis of the degradome with the CLIP-CHIP microarray.

Methods 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 PDF

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 PDF

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 PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!