Microarray of pancreatic cancer and healthy tissue
ABSTRACT: Gene expression analyses of pancreatic adenocarcinoma and adjacent ductal epithelia from the same patient using bulk vs LCM dissected samples. Our results indicate that laser capture microdissection (LCM) is necessary to identify differentially expressed genes that discriminate between PDAC and healthy pancreatic ductal tissue. Pancreatic tissues were collected at time of surgery and snap frozen in liquid nitrogen for RNA extraction and Affymetrix GeneChip Expression analyses.
Project description:We investigated the transcriptome of dentate gyrus (DG) granule cells in postmortem hippocampus from 79 subjects with mental illness (schizophrenia, bipolar disorder, major depression) or non-psychiatric controls. Material for RNA-seq analysis was harvested from tissue slides using laser capture microdissection (LCM) and aRNA amplification. Equimolar amounts of triplicate aRNA samples for each of the 79 subjects were then pooled for the preparation of sequencing libraries. Sequencing libraries were prepared using Applied Biosystem's Total RNA Sequencing Kit, following the directions for Whole Transcriptome Libraries, and analyzed with an Applied Biosystems SOLiD 4 high-throughput sequencer. We compared the performance of different normalization methods (length normalization vs. noise reductin scaling), and assembled evidence for dysruption of signaling by the micro RNA miR-182 in subjects with major depression and schizophrenia.
Project description:Cholangiocellular carcinoma (CCC) and pancreatic ductal adenocarcinoma (PDAC) are two highly aggressive cancer types which arise from epithelial cells of the pancreatobiliary system. Due to their histologic and morphologic similarity, differential diagnosis between CCC and metastasis of PDAC located in the liver frequently proves an unsolvable issue for pathologists. Biomarkers with high specificity and sensitivity for the differentiation of these tumour types would therefore be a valuable tool, but so far none are available. Here, we address this problem by comparing microdissected CCC and PDAC tumour cells in a label-free proteomics approach. The novel biomarker candidates were subsequently verified by immunohistochemical staining of primary and secondary tumour tissue.
Project description:We recently demonstrated that Msx genes, which encode muscle segment homeobox (Msh) transcription factors, regulate the transition of the luminal epithelium from high to low apicobasal polarity that is critical for implantation. In Msx deficient uteri (Msx1d/d/Msx2d/d), apicobasal polarity remains high and implantation fails to occur. However, information on the underlying molecular mechanism of Msx-dependent regulation of epithelial polarity, and the nature of epithelial-mesenchymal interactions that are characteristic of Msx genes, remain limited. In this study, we analyzed gene expression by RNA-sequencing in the luminal epithelium and stroma isolated by laser capture microdissection (LCM) on day 4 of pseudopregnancy in Msx1f/f/Msx2f/f and Msx1d/d/Msx2d/d uteri. We found upregulation of extracellular matrix components in the stroma and downregulation of immunity-related genes in both the luminal epithelium and stroma isolated from Msx1d/d/Msx2d/d mice. In addition, tight junction protein Claudin 1 and small proline-rich protein (Sprr2) were substantially upregulated in Msx1d/d/Msx2d/d epithelia. mRNA profiles of luminal epithelium and stroma from a wildtype Msx1f/f/Msx2f/f and a uterine-specific knockout Msx1d/d/Msx2d/d mouse were asessed by RNA-sequencing with the Illumina HiSeq 1500 system
Project description:LCM was perfomed on adjacent tumor and stromal cells to identify differentially expressed genes in triple negative breast cancer. To determine differences in tumor and adjacent stromal tissue, laser-capture microdissction was perfomed on 10 triple negative breast cancer specimans to isolate tumor and stromal cells for gene expression analysis. RNA was isolated from captured cells and hybridized to affymetrix gene expression microarrays.
Project description:MicroRNAs (miRNAs) are short (~22 nucleotides) regulatory RNAs that can modulate gene expression and are aberrantly expressed in many diseases including cancer. We report the results of a systems analysis of miRNA regulation in ovarian cancer. We found that 33 miRNAs are up-regulated and 9 down-regulated in CEPI relative to OSE (p<0.01, ≥2 fold change). Of these, 12 were previously annotated miRNAs (Sanger miRBase) of which 9 are up-regulated and 3 are down-regulated in CEPI relative to OSE. Current models predict that changes in levels of miRNAs will be inversely correlated with changes in the levels of targeted mRNAs due to miRNA regulation. This predicted inverse correlation held for only ~9% of predicted target mRNAs. Computational analyses indicate the unexpected low inverse correlation may be at least partially explained by variation in the number of miRNA binding sites within the 3’ UTRs of targeted mRNAs and by miRNA-mediated changes in levels of transcription factors that can exert overriding trans-regulatory controls on target loci. miRNAs were collected from three laser captured microdissected ovarian cancer epithelial (CEPI) samples. The miRNA expression pattern was compared with three healthy ovarian surface epithelia samples as controls using a custom-manufactured Affymetrix GeneChip® array.
Project description:We aimed to analyze the transcriptional profile of lung epithelial cells early after the expression of a resident K-RasG12V oncogene. This approach was based on the rationale that valuable therapeutic targets should be easier to detect in the first stages of tumor development due to tumor heterogeneity which occurr at late stages. We used a GEM model of lung adenocarcinoma driven by a resident K-RasG12V oncogene. In this GEM, the K-RasG12V allele also expresses a color marker that allows identification of K-RasG12V expressing cells at the single cell level in tissue sections. Thanks to this strategy we isolated early K-RasG12V-driven hyperplastic lesions (about 500 cells) and analyzed their gene expression profiling by Affymetrix GeneChip hybridization. Normal cells were isolated accordingly by LCM in the same lung sections.
Project description:To identify molecular changes underlying the chemopreventive or tumor promoting effects of SRD5A inhibition, we profiled gene expression changes in benign prostate epithelium from patients with PCa treated with dutasteride, a dual SRD5A inhibitor. Subjects were aged 45-80 years with clinically localized PCa (T1C to T2b), Gleason score <7, and serum PSA 2.5-10 ng/dL. 81 men were randomized to immediate RP (n=25) or to dutasterdie at 0.5 mg (n=26) or 3.5 mg (n=24) orally per day for four months prior to RP. Prostate samples embedded in OCT were used for laser capture microdissection (LCM). Keywords: clinical trial, Dutasteride, PEDB, dose response, prostate cancer, microarray Subjects were aged 45-80 years with clinically localized PCa (T1C to T2b), Gleason score <7, and serum PSA 2.5-10 ng/dL. 81 men were randomized to immediate RP (n=25) or to dutasterdie at 0.5 mg (n=26) or 3.5 mg (n=24) orally per day for four months prior to RP. Prostate samples embedded in OCT were used for laser capture microdissection (LCM). Approximately 2000-3000 epithelial cells per sample were collected from 8µm sections using the Arcturus Veritas™ Laser Capture Microdissection System according to the Arcturus HistoGene LCM Frozen Section Staining Kit Protocol (Mountain View, CA). Total RNA was isolated using the Arcturus Picopure™ Kit, and the samples were treated with DNAse using the Qiagen RNase-Free DNase Set (Qiagen Inc, Valencia, CA). Total RNA was subjected to two rounds of linear amplification using the Ambion MessageAmpII Kit (Ambion Inc, Austin, TX), quantitated in a Gene-Spec III spectrophotometer (Hitachi, Tokyo) and aRNA integrity evaluated using gel electrophoresis. 40 Human Prostate (PEDB) Microarrays were run with sample on the Cy3 channel against a Human Gold Standard (v5) on the Cy5 channel.
Project description:MicroRNAs (miRNAs) are short (~22 nucleotides) regulatory RNAs that can modulate gene expression and are aberrantly expressed in many diseases including cancer. We report the results of a systems analysis of miRNA regulation in ovarian cancer. We found that 33 miRNAs are up-regulated and 9 down-regulated in CEPI relative to OSE (p<0.01, ≥2 fold change). Of these, 12 were previously annotated miRNAs (Sanger miRBase) of which 9 are up-regulated and 3 are down-regulated in CEPI relative to OSE. Current models predict that changes in levels of miRNAs will be inversely correlated with changes in the levels of targeted mRNAs due to miRNA regulation. This predicted inverse correlation held for only ~9% of predicted target mRNAs. Computational analyses indicate the unexpected low inverse correlation may be at least partially explained by variation in the number of miRNA binding sites within the 3’ UTRs of targeted mRNAs and by miRNA-mediated changes in levels of transcription factors that can exert overriding trans-regulatory controls on target loci. mRNAs were collected from three laser captured microdissected ovarian cancer epithelial (CEPI) samples. The mRNA expression pattern was compared with five healthy ovarian surface epithelia samples as controls using the Affymetrix U133 Plus 2.0 3' expression array.
Project description:Transforming Growth Factor Beta-induced (TGFBI)-related dystrophies constitute the most common heritable forms of corneal dystrophy worldwide. However, other than the underlying genotypes of these conditions, a limited knowledge exists of the exact pathomechanisms of these disorders. This study expands on our previous research investigating dystrophic stromal aggregates, with the aim of better elucidating the pathomechanism of 2 conditions arising from the most common TGFBI mutations: granular corneal dystrophy (GCD1; R555W), and lattice corneal dystrophy (LCD1; R124C). GCD1 and LCD1 patient corneas were stained with H&E and Congo red to visualise stromal non-amyloid and amyloid deposits, respectively. Laser capture microdissection was used to isolate aggregates and extracted protein was analyzed by mass spectrometry. Proteins were identified and their approximate abundances were determined. Spectra of TGFBIp peptides were also recorded and quantified. In total, 3 proteins were found within GCD1 aggregates that were absent in the healthy control corneal tissue. In comparison an additional 18 and 24 proteins within stromal LCD1 and Bowman’s LCD1 deposits, respectively, were identified. Variances surrounding the endogenous cleavage sites of TGFBIp were also noted. An increase in the number of residues experiencing cleavage was observed in both GCD1 aggregates and LCD1 deposits. The study reveals previously unknown differences 1 between the protein composition of GCD1 and LCD1 aggregates, and confirms the presence of the HtrA1 protease in LCD1-amyloid aggregates. In addition, we find mutation specific differences in the processingof mutant TGFBIp species, which may contribute to the variable phenotypes noted in TGFBI-related dystrophies.
Project description:Intrahepatic cholangiocarcinoma (ICC) is the second most common type of primary cancer in the liver. ICC is an aggressive cancer with poor prognosis and limited therapeutic strategies. The identification of new drug targets and prognostic biomarkers is an important clinical challenge for ICC. The presence of an abundant stroma is a histological hallmark of ICC. Given the well established role of the stromal compartment in the progression of cancer diseases, we hypothesized that relevant biomarkers could be identified by analyzing the stroma of ICC. By combining laser capture microdissection and gene expression profiling we demonstrated that ICC stromal cells exhibit dramatic genomic changes. We identified a signature of 1,073 non-redundant genes that significantly discriminate the tumor stroma from non tumor fibrous tissue. Functional analysis of differentially expressed genes demonstrated that up-regulated genes in the stroma of ICC were related to cell cycle, extracellular matrix and Transforming Growth Factor beta (TGFβ) pathways. Tissue microarray analysis using an independent cohort of 40 ICC patients validated at a protein level the increase expression of Collagen 4, Laminin, Osteopontin/SPP1, KIAA0101 and TGFβ2 genes in the stroma of ICC. Statistical analysis of clinical and pathological features demonstrated that the expression of Osteopontin, TGFβ2 and Laminin in the stroma of ICC was significantly correlated with patient overall survival. More importantly, multivariate analysis demonstrated that the stromal expression of Osteopontin was an independent prognostic marker for overall and disease-free survival. Conclusion: The study identifies clinically relevant genomic alterations in the stroma of ICC, including candidates biomarkers for prognosis, supporting the idea that tumor stroma is an important factor for ICC onset and progression. 20 RNA samples were analyzed, from 10 patients with ICC. For each patient, RNA were isolated from laser capture microdissected (LCM) stroma from tumor and non tumor areas.