ABSTRACT: Frozen blocks of human embryonic limb tissue were sectioned and laser capture microdissected to collect three human chodrocyte tissues - precursor, differentiated and hypertrophic chondrocytes.Total RNA including miRNAs was isolated and analyzed using Applied Biosystems OpenArrayM-BM-. Real-Time PCR system. RT-PCR miRNA expression profiling. miRNA expression from three chondrocyte sub types - precursor, differentiated and hypertrophic chondrocytes were analyzed using TaqManM-BM-. OpenArrayM-BM-. Human MicroRNA Panel.
Project description:In this study, we compared the treatment with one of 2 GLP-1 receptor agonists, Liraglutide and Semaglutide on the gene expression in 6 different DIO rat brain areas known to express the GLP-1 receptor. DIO rats were treated with vehicle, liraglutide, semaglutide or weight-matched for 23 days and tissue from the brain areas LS, PVH, ARH, DMH, AP and NTS was obtained with LCM.
Project description:The Hos: HR-1 mouse strain (HR-1) is an autosomal resessive mutant strain which is widely used in pharmacological and dermatological studies in Japan. Although HR-1 mice have been believed to be Hr gene mutants based on their distinctive alopecia phenotype, there are no reports on either the genetic nature of the Hr gene or mechanism of alopecia. The present study clarified that the HR-1 mice carry a C to T transition in the Hr gene, which causes a proline to serin amino acid change in the functional domain of translated HR protein. It is well known that HR plays an important role in hair follicle regression, and loss of HR function triggers distinctive alopecia with the presence of premature apoptosis in the regressing hair follicle. However, the detailed function of HR protein is still uncertain. To clarify the mechanism by which HR dysfunction causes hair loss, here we performed microarray analysis on the mRNA samples obtained from the regressing hair follicles of the homozygous (HrHos / HrHos) and heterozygous (+/HrHos, control) Hr mutant mice using laser capture microdissection. Our results showed increased expression of apoptosis-related and cell-cycle-repressive genes and decreased expression of cell-cycle-promoting genes, suggesting inhibition of the cell cycle at G0/G1. These findings strongly suggest that the loss of HR function leads to high expression of cell cycle inhibitors, resulting in the hair loss with premature apoptosis in the hair matrix. Experiment Overall Design: Homozygous (HrHos / HrHos) and heterozygous (+/HrHos) Hr mutant mice of the same N1 littermates obtained from a male Hos: HR-1 mouse and a female heterozygous F1 offspring (+/HrHos) between an HR-1 mouse and an ICR mouse were used at day 14 postpartum. +/HrHos mice were used as controls because the Hr gene mutantion is autosomal recessive and the morphological appearance of +/HrHos is as same as that of wild-type mice. Four samples (two from +/HrHos and two from HrHos / HrHos) were analyzed. All RNA samples were extracted from hair follicles of dorsal skin using Laser capture microdissection.
Project description:Patients diagnosed with breast cancer were recruited to an important trial, the Tamoxifen Window Trial, in two series, series A during 1988-1989 and series B during 1989-1990. Some patients received tamoxifen for short windows. Clinical samples (biopsies) for this trial were collected before and after treatment. These biopsies were available as formalin-fixed paraffin-embedded (FFPE) tissue blocks. Responding patients and non-responding patients were determined by the change in the Ki67 scores. Responding patients were identified as those that had demonstrated a reduction in their Ki67 scores, and non-responding patients were identified as those in which Ki67 had not changed or had increased. The change in the percentage of Ki67 positive tumour cells was obtained by comparing the pre-treatment and post-treatment scores and calculating the difference in the scores. Laser capture microdissection (LCM) was utilised to microdissect the epithelial and stromal cells from the post-treatment breast cancer FFPE tissue sections of responding and non-responding patients. Samples obtained following LCM underwent RNA extraction and were subsequently used for gene expression profiling. Post-treatment samples from three responding patients (A29, B42, B61) and three non-responding patients (A15, A19, B59) were identified as suitable for this study and underwent LCM. Samples were analysed using Affymetrix GeneChipM-BM-. Human Exon 1.0 ST array. No technical replicates were performed. The stromal compartments collected from post-treatment samples of patients B59 and B61 failed to generate the appropriate yield of cDNA following two rounds of amplification and consequently were excluded from further analysis. Array hybridisation of samples were performed in 3 separate batches.
Project description:Ductal carcinoma in situ (DCIS) is a precursor lesion that can give rise to invasive breast cancer (IBC). It has been proposed that both the nature of the lesion and the tumor microenvironment play key roles in progression to IBC. Here, laser capture microdissected tissue samples from epithelium and stroma in normal breast, pure DCIS, and pure IBC were employed to define key gene expression profiles associated with disease progression. Tumor and matching stroma were profiled for 9 DCIS patients, 10 IBC patients, and 3 normal breast. Differential gene expression was evaluated for paired normal stroma versus normal epitelium samples, paired DCIS stroma versus DCIS epitelium samples, paired IBC stroma versus IBC epitelium, IBC stroma versus DCIS stroma, and IBC epithelium versus DCIS epithelium.
Project description:The breast tumor microenvironment plays an active role in tumorigenesis. Molecular alterations, including epigenetic modifications to DNA, and changes in RNA and protein expression have been identified in tumor-associated stroma; however, there is considerable debate as to whether the stroma is characterized by genomic instability or whether detection of chromosomal alterations in the breast stroma is a reflection of technological artifact rather than the true genomic content of the tumor microenvironment. Methods: Surgically-removed breast stroma specimens from 112 women undergoing reductive mammoplasty (n=15), prophylactic mastectomy (N=6) or mastectomy for a diagnosis of breast disease (n=92) were frozen in optimal cutting temperature medium. Allelic imbalance (AI) analysis was performed in 484 stromal specimens from 98 women using a panel of 52 microsatellite markers; SNP data was generated from a subset of 86 stromal specimens using 250K SNP arrays (Affymetrix). Copy number alterations were identified using Partek Genomics Suite. Results: AI was not detected in 92% (444/484) of stroma specimens. When compared to previously generated AI data from 77 formalin-fixed, paraffin-embedded stroma specimens (Ellsworth et al., Ann Surg Oncol 2004), 32 (42%) of which harbored at least one detectable AI event, the frequency of AI in the FFPE specimens (4.62%) was significantly higher (P<0.0001) than that found in frozen specimens (0.45%). Of the stroma specimens assayed using SNP arrays 95% (82/86) had no detectable alterations and the 11 copy number changes were small and not shared between specimens. Conclusions: The data presented here support a model in which the tumor microenvironment is genetically stable. The direct comparison of copy number alterations between FFPE and frozen research-grade specimens using identical methodologies suggests that past reports of significant AI in breast stroma, both adjacent to and distant from the tumor, reflects artifact in the archival specimens caused by formalin-fixation, paraffin-embedding and tissue storage. 250K STY data was generated for 86 breast stromal specimens. The tissue specimens were analyzed by paired analysis to the SNP data from matched genomic (blood) DNAs
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 (TGFM-NM-2) 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 TGFM-NM-22 genes in the stroma of ICC. Statistical analysis of clinical and pathological features demonstrated that the expression of Osteopontin, TGFM-NM-22 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.
Project description:To investigate the impact of ablating Bcl9/Bcl9l on tumorigenesis, 6-8- week-old mice were exposed first to a single dose dimethylhydrazine (DMH, 44 mg/kg body weight), which is metabolized in the liver to carcinogenic azoxymethane (AOM), followed by 7 days oral administration of 2 % dextrane sulfate sodium (DSS) in the drinking water. This regimen results in the emergence of dysplastic adenomas, which progress to differentiated adenocarcinomas that are morphologically similar to human colorectal adenocarcinomas and typically harbor β-catenin stabilizing mutations of GSK3ß phosphorylation sites. Accordingly, these tumors present hallmarks of active Wnt signaling such as accumulation of nuclear β-catenin and expression of Wnt target genes. Total RNA of laser dissected samples from five different tumors each of two wild-type mice and three vil-Cre-Bcl9-/-/Bcl9l-/- mice was collected and resulting amplified cDNA hybridized to Affymetrix Mouse Genome 430 2.0 arrays. Samples are labeled as follows: Genotype_TumorID_MouseID_UniqueID.
Project description:We set out to generate transcriptional maps of chondrocyte UPR gene networks in vivo using two mouse models (Schmid and Cog) of Schmid chondrodysplasia, in order to define the consequences of UPR activation for the adaptation, differentiation, and survival of chondrocytes experiencing ER stress during hypertrophy, thus providing insights into ER stress signaling and its impact on cartilage pathophysiology. Our data demonstrate that both models displayed similar unfolded protein responses (UPRs), involving activation of ER stress sensors Ire1 and Atf6 and upregulation of their downstream targets, including molecular chaperones, foldases, and ER-associated degradation machinery. Also upregulated were the emerging UPR regulators Wfs1 and Syvn1, recently identified UPR components including Armet and Creld2, and genes not previously implicated in ER stress such as Steap1 and Fgf21. Moreover, we transcriptionally profiled the expression of wildtype growth plate zone gene signatures in the mutant hypertrophic zones, in order to define the differentiation status of ER-stressed chondrocytes in the mutant hypertrophic zones. Hypertrophic zone gene upregulation and proliferative zone gene downregulation were both inhibited in Schmid hypertrophic zones, resulting in the persistence of a proliferative chondrocyte-like expression profile in ER-stressed Schmid chondrocytes. For the mutant hypertrophic zone gene expression profiling, the hypertrophic zone from one tibia from each of three two week old Schmid, wildtype (Schmid background), Cog, and wildtype (Cog background) mice was microdissected. In all cases, total RNA was extracted and amplified through two rounds of linear amplification, labelled with Cy3, and interrogated by microarray analysis using the Agilent 44K, mouse whole genome platform.
Project description:Postnatal neuroblasts were microdissected by laser micro dissection from posterior and anterior rostral migratory stream (RMS). Neuroblasts in the posterior RMS are just started their migration. Neuroblasts in the anterior RMS are already migrated for several days in the stream