The use of microarrays and fluorescence in situ hybridization for the study of mechanotransduction from topography.
The combination of transcriptomic analysis and fluorescence in situ hybridization (FISH) provides a robust methodology to study genomic changes in different biological conditions. Microarrays allow a global study of gene expression in response to the conditions of interest, with comparison between control(s) and one or more test condition(s). The messenger RNA amplification step permits detection of even low abundance transcripts, a critical advantage for applications such as biomaterials research, where the starting material may be limited. Different types of microarrays are commercially available that allow the investigation of specific features, such as exon arrays, microRNA arrays, and gene arrays. Microarrays are available for different model organisms, but we use Affymetrix ® HuGene ® ST (Sense Target) arrays, a type of gene array for analysis of human samples. FISH involves fluorescent detection of probe DNA hybridized to an in situ chromosomal target that can be either whole chromosomes or chromosomal segments. The overall hybridization is similar to labeling with radioactive probes but the incorporation of fluorescent detection of the probe sequences allows for high sensitivity in a simple and quick assay. FISH can be applied to a variety of specimen types depending on the study of interest. In this chapter, we describe the methodologies of these two techniques and provide technical tips that should help overcome challenges in carrying them out.
Centre for Cell Engineering, Institute of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Laura E
LE
McNamara
Centre for Cell Engineering, Institute of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Matthew J
MJ
Dalby
Centre for Cell Engineering, Institute of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Monica P
MP
Tsimbouri
D002875:Chromosomes / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D040542:Mechanotransduction, Cellular; D046228:Microarray Analysis / Q000379:methods*
D015342:DNA Probes
Chromosome; Fluorescence in situ hybridization; Gene expression; Microarrays; Microscopy; RNA