Expression profiling of microdissected cell populations selected from basal cells in normal epidermis and basal cell carcinoma
ABSTRACT Basal cell carcinomas (BCCs) are prevalent tumours with uniform histology that develop without any known precursor lesion. Alterations in the sonic hedgehog-patched1 signalling pathway are accepted as necessary events for tumorigenesis, and mutations in the patched1 gene are frequently present in tumours.
To analyse transcript profiles in BCC.
We used laser-assisted microdissection to isolate and collect cell populations defined under the microscope. Peripheral cells from nests of BCC were selected to represent tumour cells, and normal keratinocytes from epidermis basal layer were used as control. Extracted RNA was amplified and hybridized on to a cDNA microarray. Results Our results show that BCC cells express a transcript signature that is significantly different from that of normal keratinocytes, and over 350 genes with various functions were identified as differentially expressed. The compiled data suggest an upregulation of the Wnt signalling pathway as a major event in BCC cells. Furthermore, tumour cells appear to have an increased sensitivity to oxygen radicals and dysregulated genes involved in antigen presentation.
were validated at both the transcriptional level using real-time polymerase chain reaction and at the protein level using immunohistochemistry.
We show that microdissection in combination with robust strategies for RNA extraction, amplification and cDNA microarray analysis allow for reliable transcript profiling and that antibody-based proteomics provides an advantageous strategy for the analysis of corresponding differentially expressed proteins. We found that expression patterns were significantly altered in BCC cells compared with basal keratinocytes and that the Wnt signalling pathway was upregulated in tumour cells.
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ABSTRACT: The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at -80[degree sign]C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 mum2 in LEC to 392,887 mum2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/mul. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 mul. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 mum2 to 130,0000 mum2. RNA concentration of these samples ranged from 10.88 ng/12 mul to 25.8 ng/12 mul, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded. The optimised protocol for sample collection and laser microdissection improved the RNA yield of the insitu ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms.BMC Ophthalmology 10/2013; 13(1):62. DOI:10.1186/1471-2415-13-62 · 1.08 Impact Factor
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ABSTRACT: Merkel cell carcinoma (MCC) is a rare and aggressive type of skin cancer that is predominantly caused by infection by the Merkel cell polyomavirus. The cell of origin for MCC is still debated because conflicting data suggest that MCC cells could be derived from Merkel cells (MCs)  or their precursors . aggressive carcinoma characteristically occurs on sun-exposed areas of elderly white and immunosupressed patients, with approximately 50% of all tumors occuring on the face and neck and 40% in the extremities .This article is protected by copyright. All rights reserved.Experimental Dermatology 09/2014; 23(12). DOI:10.1111/exd.12546 · 4.12 Impact Factor
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ABSTRACT: Gene expression profiling can be useful for phenotypic classification, investigation of functional pathways, and to facilitate the search for disease risk genes through the integration of transcriptional data with available genomic information. To enhance our understanding of the genetic and molecular basis of basal cell carcinoma (BCC) we performed global gene expression analysis to generate a disease-associated transcriptional profile. A gene signature composed of 331 differentially expressed genes (DEGs) was generated from comparing 4 lesional and 4 site-matched control samples using Affymetrix Human Genome U95A microarrays. Hierarchical clustering based on the obtained gene signature separated the samples into their corresponding phenotype. Pathway analysis identified several significantly overrepresented pathways including PPAR-γ signaling, TGF-β signaling and lipid metabolism, as well as confirmed the importance of SHH and p53 in the pathogenesis of BCC. Comparison of our microarray data with previous microarray studies revealed 13 DEGs overlapping in 3 studies. Several of these overlapping genes function in lipid metabolism or are components of the extracellular matrix, suggesting the importance of these and related pathways in BCC pathogenesis. BCC-associated DEGs were mapped to previously reported BCC susceptibility loci including 1p36, 1q42, 5p13.3, 5p15 and 12q11-13. Our analysis also revealed transcriptional 'hot spots' on chromosome 5 which help to confirm (5p13 and 5p15) and suggest novel (5q11.2-14.3, 5q22.1-23.3 and 5q31-35.3) disease susceptibility loci/regions. Integrating microarray analyses with reported genetic information helps to confirm and suggest novel disease susceptibility loci/regions. Identification of these specific genomic and/or transcriptional targets may lead to novel diagnostic and therapeutic modalities.International Journal of Oncology 11/2012; 42(2). DOI:10.3892/ijo.2012.1725 · 2.77 Impact Factor