Use of High Throughput Microarray Membranes for cDNA Analysis of Cutaneous Wound Repair

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Nicole S. Gibran and F. Frank Isik 1. Introduction

Hypertrophic scar formation represents an abnormal wound-healing response following thermal injuries or partial-thickness wounds. Specific growth factors, cytokines, extracellular matrix molecules, and proteinases that are known to alter cell proliferation and migration have been implicated in the generation of hypertrophic scars. However, the etiology of hypertrophic scarring has not been identified. Given the complex molecular mechanisms of wound repair, differences in expression of isolated functional genes alone may not sufficiently explain clinical variations. Other genes, such as transcriptional regulators, control response to injury and may provide a more comprehensive explanation for the different responses to injury. Gene expression by Northern blot analysis or in situ hybridization (ISH) to determine levels of mRNA in tissue samples limits the study of tissues to a single gene and requires a large amount of sample. Reverse transcriptase polymerase chain reaction (PCR) and ribonuclease protection assays allow detection of smaller amounts of mRNA in less tissue but are still restricted by the limited number of genes that can be targeted per assay and by the time involved. With the advent of cDNA microarray technology, a broad-scale evaluation of differential gene expression in hypertrophic scar formation is attainable (1-5). This technology permits simultaneous broad evaluation of previously unsuspected genes such as cell signaling and transcription genes.

Because of the ability to store large data sets and compare different experiments, it is possible not only to compare hypertropic scar with normal scar or to normal skin, as we have previously reported, but also to compare the data from those arrays to other data sets—such as acute wounds. This allows a broad-scale evaluation of events that may be comparable in an early inflamed wound and a late inflamed hypertrophic scar. This versatility may elicit differences in the early wounds that define later events in wound repair.

High-throughput cDNA microarrays provide expression analysis of thousands of genes simultaneously. Most published microarray studies have focused on isolated cells in culture, rather than on human tissues. Whereas the multiple distinct cell types in skin magnify the complexity of interpreting cDNA microarray data, the most powerful applications of cDNA microarray technology will involve analysis of complex human tissues, such as skin, in normal and disease states (6).

We describe one approach to cDNA microarray analysis using a commercially available microarray system ( genefilters; Research Genetics, Huntsville, AL) to evaluate differences in expression of 4000 known human genes in hypertrophic scar (7).

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