Project description:Analysis of cell specific gene expression in mycorrhizal rice roots. Described in Roth, Chiapello et al. 2019 We used LCM to harvest arbusculated and adjacent systemic cells from mycorrhizal rice roots, along with cortical cells from mock inoculated plants
Project description:We describe the use of laser capture microdissection (LCM) to isolate human and murine sebaceous glands (SGs) for transcriptomic analysis and publish this SG transcriptomic data for reference. We show that compared to whole skin RNA sequencing, LCM RNA sequencing allows for high resolution in identifying and describing SG genes at homeostasis. Lastly, we compare this LCM of sebaceous glands to published SG clusters from single cell RNA sequencing of skin, and show that we achieve greater resolution and depth with the LCM approach.
Project description:Atherosclerosis is a transmural chronic inflammatory condition of small and large arteries that is associated with adaptive immune responses at all disease stages. However, impacts of adaptive immune reactions on clinically apparent atherosclerosis such as intima lesion (plaque) rupture, thrombosis, myocardial infarction, and aneurysm largely remain to be identified. It is increasingly recognized that leukocyte infiltrates in plaque, media, and adventitia are distinct but their specific roles have not been defined. To map these infiltrates, we employed laser capture microdissection (LCM) to isolate the three arterial wall laminae using apoE-/- mouse aorta as a model. RNA from LCM-separated tissues was extracted and large scale whole genome expression microarrays were prepared. We observed that the quality of the resulting gene expression maps was compromised by tissue RNA carried over from adjacent laminae during LCM. To account for these flaws, we established quality controls and algorithms to improve the predictive power of LCM-derived microarray data. Our approach creates robust transcriptome atlases of normal and atherosclerotic aorta. Assessing LCM transcriptomes for immunity-related mRNAs indicated markedly distinctive gene expression patterns in the three laminae of the atherosclerotic aorta. These mouse mRNA expression data banks can now be mined to address a wide range of questions in cardiovascular biology.
Project description:We used laser-capture microdissection (LCM) to separate tumor epithelium (TE) from stroma (TS) in 23 ILC samples. Gene expression analysis was used to identify genes that are enriched in the stroma of ILC, but not IDC or normal breast.
Project description:Atherosclerosis is a transmural chronic inflammatory condition of small and large arteries that is associated with adaptive immune responses at all disease stages. However, impacts of adaptive immune reactions on clinically apparent atherosclerosis such as intima lesion (plaque) rupture, thrombosis, myocardial infarction, and aneurysm largely remain to be identified. It is increasingly recognized that leukocyte infiltrates in plaque, media, and adventitia are distinct but their specific roles have not been defined. To map these infiltrates, we employed laser capture microdissection (LCM) to isolate the three arterial wall laminae using apoE-/- mouse aorta as a model. RNA from LCM-separated tissues was extracted and large scale whole genome expression microarrays were prepared. We observed that the quality of the resulting gene expression maps was compromised by tissue RNA carried over from adjacent laminae during LCM. To account for these flaws, we established quality controls and algorithms to improve the predictive power of LCM-derived microarray data. Our approach creates robust transcriptome atlases of normal and atherosclerotic aorta. Assessing LCM transcriptomes for immunity-related mRNAs indicated markedly distinctive gene expression patterns in the three laminae of the atherosclerotic aorta. These mouse mRNA expression data banks can now be mined to address a wide range of questions in cardiovascular biology. Wild-type and apoE-deficient mice on the C57BL/6J genetic background were maintained on a standard mouse chow. Total aortae were removed at the age of 78 weeks, abdominal aorta was separated from the remainder of the tissue and arterial wall laminae were separated by laser capture microdissection as described (Beer et al. 2011). Following RNA quality controls, microarrays were prepared following MIAME guidelines as described previously (Uzonyi et al. 2006; Graebner et al. 2009; Lotzer et al. 2010).
Project description:We used laser capture microdissection (LCM) to capture soybean seed compartments and profiled the transcriptomes of several compartments using next-generation sequencing. We profiled the transcriptomes of the embryo-proper and the suspensor region from globular stage embryos and the seed coat parenchyma layer of early maturation stage seeds using the Illumina GAIIx system.
Project description:Laser Capture Microdissection (LCM) followed by RNA-seq of apical and basal domains of follicle cells from stage 9-10B Drosophila egg chambers. The experiment is composed of 5 Apical and 5 Basal biological replicates. Each biological replicate consists of a pool of 10 LCM fragments. Each fragment consists of 5-10 adjacent cell domains microdissected from either the apical half or the basal half of the follicular epithelium.
Project description:DNA methylation is a mechanism for long-term transcriptional regulation and is required for normal cellular differentiation. Failure to properly establish or maintain DNA methylation patterns leads to cell dysfunction and diseases such as cancer. Identifying DNA methylation signatures in complex tissues can be challenging due to inaccurate cell enrichment methods and low DNA yields. We have developed a technique called Laser Capture Microdissection-Reduced Representation Bisulfite Sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of CpG Islands and promoters. LCM-RRBS accurately and reproducibly profiles genome-wide methylation of DNA extracted from microdissected fresh frozen or formalin-fixed paraffin-embedded tissue samples. To demonstrate the utility of LCM-RRBS, we characterized changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Compared to adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development. LCM-RRBS is a rapid, cost-effective, and sensitive technique for analyzing DNA methylation in heterogeneous tissues and will facilitate the investigation of DNA methylation in cancer and organ development. Laser capture microdissection-reduced representation bisulfite sequencing and reduced representation bisulfite sequencing on human blood leukocyte, human endometrial tumor, mouse liver tissue, and mouse normal and neoplastic adrenal tissue
Project description:The goal of the study was to characterize the whole genome transcriptome profiles of pure sample population of human ameloblastoma epithelial cells for examination of molecular pathways. Seventeen human ameloblastoma samples were obtained. Both fresh frozen and FFPE samples were used and placed in solution for 1-4 weeks to allow decalcification by EDTA. The tissue was sectioned at -35C at a thickness of 7 microns. These sections were used for laser capture microdissection (LCM) to isolate the neoplastic epithelial portion of the tumors, using static image settings. Total RNA was extracted from each sample and examined using the whole genome microarray against the Stratagene universal human reference RNA.
Project description:DNA methylation is a mechanism for long-term transcriptional regulation and is required for normal cellular differentiation. Failure to properly establish or maintain DNA methylation patterns leads to cell dysfunction and diseases such as cancer. Identifying DNA methylation signatures in complex tissues can be challenging due to inaccurate cell enrichment methods and low DNA yields. We have developed a technique called Laser Capture Microdissection-Reduced Representation Bisulfite Sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of CpG Islands and promoters. LCM-RRBS accurately and reproducibly profiles genome-wide methylation of DNA extracted from microdissected fresh frozen or formalin-fixed paraffin-embedded tissue samples. To demonstrate the utility of LCM-RRBS, we characterized changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Compared to adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development. LCM-RRBS is a rapid, cost-effective, and sensitive technique for analyzing DNA methylation in heterogeneous tissues and will facilitate the investigation of DNA methylation in cancer and organ development.