Expression profiling of microdissected cell populations selected from basal cells in normal epidermis and basal cell carcinoma
Department of Genetics and Pathology, Uppsala University Hospital, SE-751 85 Uppsala, Sweden. British Journal of Dermatology
(Impact Factor: 4.28).
04/2008; 158(3):527-38. DOI: 10.1111/j.1365-2133.2007.08418.x
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.
Available from: Harminder Dua
- "A study on laser microdissected breast tissue samples by Cowherd et al. in 2004 showed that the technical variability introduced by amplification and hybridisation experiments is smaller than the actual biological variation for different types of breast cancer tissues demonstrated with differential gene expression in these samples . Samples for transcriptional profiling studies on epidermal basal cells, endometriosis tissue, and osteocytes were successfully collected with “Positioning and Ablation in Laser Microdissection” (PALM®) microbeam systems (PALM Microlaser Technologies GmbH) . This technique grew in popularity as it allowed precise dissection of tissues under direct visualisation by a non-contact technique. "
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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.
Available from: Zhengqing Ouyang
- "In some studies of mRNA authors identified genes whose expression was different in normal skin vs. basal cell tumors (Asplund et al. 2008; O'Driscoll et al. 2006), but the normal cell of origin with which BCC tissue should be compared is probably a hair follicle stem cell (Sellheyer 2011), and therefore these expression differences may not be specific to tumorigenesis. The ability to distinguish histopathologic subtypes was limited. "
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ABSTRACT: Basal cell carcinomas (BCCs) are the most common cancers in the United States. The histologic appearance distinguishes several subtypes, each of which can have a different biologic behavior. In this study, global miRNA expression was quantified by high-throughput sequencing in nodular BCCs, a subtype that is slow growing, and infiltrative BCCs, aggressive tumors that extend through the dermis and invade structures such as cutaneous nerves. Principal components analysis correctly classified seven of eight infiltrative tumors on the basis of miRNA expression. The remaining tumor, on pathology review, contained a mixture of nodular and infiltrative elements. Nodular tumors did not cluster tightly, likely reflecting broader histopathologic diversity in this class, but trended toward forming a group separate from infiltrative BCCs. Quantitative polymerase chain reaction assays were developed for six of the miRNAs that showed significant differences between the BCC subtypes, and five of these six were validated in a replication set of four infiltrative and three nodular tumors. The expression level of miR-183, a miRNA that inhibits invasion and metastasis in several types of malignancies, was consistently lower in infiltrative than nodular tumors and could be one element underlying the difference in invasiveness. These results represent the first miRNA profiling study in BCCs and demonstrate that miRNA gene expression may be involved in tumor pathogenesis and particularly in determining the aggressiveness of these malignancies.
Available from: Anna Asplund
- "The underlying assumption is that differences in mRNA levels are manifested in different phenotypes as a result of differences in protein levels. Accordingly, correlations between the differential expression of specific mRNAs and corresponding proteins have been found in numerous studies , many of which have been shown to have clear biological relevance [2,3]. Several studies have also found significant general correlations between RNA levels and protein levels [4-10], usually using data on RNA abundance acquired from platforms such as microarrays and Serial Analysis of Gene Expression (SAGE), in conjunction with data on the abundance of corresponding proteins derived from mass spectrometry (MS) analyses. "
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ABSTRACT: The Central Dogma of biology holds, in famously simplified terms, that DNA makes RNA makes proteins, but there is considerable uncertainty regarding the general, genome-wide correlation between levels of RNA and corresponding proteins. Therefore, to assess degrees of this correlation we compared the RNA profiles (determined using both cDNA- and oligo-based microarrays) and protein profiles (determined immunohistochemically in tissue microarrays) of 1066 gene products in 23 human cell lines.
A high mean correlation coefficient (0.52) was obtained from the pairwise comparison of RNA levels determined by the two platforms. Significant correlations, with correlation coefficients exceeding 0.445, between protein and RNA levels were also obtained for a third of the specific gene products. However, the correlation coefficients between levels of RNA and protein products of specific genes varied widely, and the mean correlations between the protein and corresponding RNA levels determined using the cDNA- and oligo-based microarrays were 0.25 and 0.20, respectively.
Significant correlations were found in one third of the examined RNA species and corresponding proteins. These results suggest that RNA profiling might provide indirect support to antibodies' specificity, since whenever a evident correlation between the RNA and protein profiles exists, this can sustain that the antibodies used in the immunoassay recognized their cognate antigens.
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