MACE-sequencing of carotid neointimal tissue isolated by laser capture microdissection following carotid wire injury in mice
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE220512 | GEO | 2023/03/09
REPOSITORIES: GEO
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Project description:MACE-sequencing of carotid neointimal tissue isolated by laser capture microdissection following carotid wire injury in mice
| PRJNA910101 | ENA
Project description:IRF9 is ubiquitously expressed and mediates the effects of IFNs, previous study showed that IRF9 played an important role in immunity and cell fate decision. Our recent study revealed that IRF9 involved in cardiac hypertrophy, hepatic steatosis and insulin resistance. However, the function of IRF9 in VSMC and neointima formation was largely unknown. We found that IRF9 expression was significantly increased in the VSMCs of mouse carotid artery. More importantly, we generated SMC-specific IRF9 overexpression transgenic mice (IRF9 TG) and found that IRF9 TG significantly increased VSMC proliferation, migration and neointima formation compared with NTG mice in response to injury. To evaluate the underlying mechanism by which IRF9 promotes VSMC proliferation and migration after vascular injury, IRF9 TG and NTG mice were subjected to wire-injury and the carotid arteries were collected at 14 days post-injury. We combined 3-5 vessels for one sample, and 3 samples for each phenotype. Subsequently, a total of 400ng RNA was used following Affymetrix instruction and 10 ug of cRNA were hybridized for 16 hr at 45°. GeneChips were scanned using the Scanner 7G and the data was analyzed with Expression Console using Affymetrix default analysis settings and global scaling as normalization method. RMA analysis was employed to evaluate the gene expression. We used microarrays to detect the global gene expression in the carotid arteries of smooth muscle cell specific IRF9 transgenic mice(IRF9 TG) compared with non transgenic control mice (NTG) at 14 days post-injury and identified distinct classes of altered genes. non-transgenic controls mice (NTG) and smooth muscle specific IRF9 transgenic mice (IRF9 TG) were subjected to wire-injury and the carotid ateries were collected at 14 days post-injury. We combine 3-5 vessels in one tube and for a single Affymetrix microarray. Total RNA was extracted and a total of 400ng RNA was used following Affymetrix instruction. 3 biological samples for each genotype.
2014-09-03 | E-GEOD-61046 | biostudies-arrayexpress
Project description:IRF9 is ubiquitously expressed and mediates the effects of IFNs, previous study showed that IRF9 played an important role in immunity and cell fate decision. Our recent study revealed that IRF9 involved in cardiac hypertrophy, hepatic steatosis and insulin resistance. However, the function of IRF9 in VSMC and neointima formation was largely unknown. We found that IRF9 expression was significantly increased in the VSMCs of mouse carotid artery. More importantly, we generated SMC-specific IRF9 overexpression transgenic mice (IRF9 TG) and found that IRF9 TG significantly increased VSMC proliferation, migration and neointima formation compared with NTG mice in response to injury. To evaluate the underlying mechanism by which IRF9 promotes VSMC proliferation and migration after vascular injury, IRF9 TG and NTG mice were subjected to wire-injury and the carotid arteries were collected at 14 days post-injury. We combined 3-5 vessels for one sample, and 3 samples for each phenotype. Subsequently, a total of 400ng RNA was used following Affymetrix instruction and 10 ug of cRNA were hybridized for 16 hr at 45°. GeneChips were scanned using the Scanner 7G and the data was analyzed with Expression Console using Affymetrix default analysis settings and global scaling as normalization method. RMA analysis was employed to evaluate the gene expression. We used microarrays to detect the global gene expression in the carotid arteries of smooth muscle cell specific IRF9 transgenic mice(IRF9 TG) compared with non transgenic control mice (NTG) at 14 days post-injury and identified distinct classes of altered genes.
2014-09-03 | GSE61046 | GEO
Project description:IRF4 is mainly expressed in immune cells, including B cell, T cell, macrophage and dendritic cell. Previous study showed that IRF4 plays important roles in regulating the differentiation and maturation of immune cells. Recently, our and other`s studies revealed that IRF4 involved in the pathogenesis of cardiac hypertrophy, cerebral ischemic reperfusion injury and metabolic disorder. However, the function of IRF4 in VSMC and neointima formation was largely unknown. We found that IRF4 expression was dramatically decreased in the VSMCs of mouse carotid artery. More importantly, using global IRF4 deficient mouse (KO), we demonstrated that IRF4 deficiency significantly increased VSMC proliferation, migration and neointima formation compared with wild type mice (WT) in response to injury. To evaluate the underlying mechanism by which IRF4 promotes VSMC proliferation and migration after vascular injury, IRF4 KO and WT mice were subjected to wire-injury and the carotid arteries were collected at 14 days post-injury. We combined 3-5 vessels for one sample, and 3 samples for each phenotype. Subsequently, a total of 400ng RNA was used following Affymetrix instruction and 10 ug of cRNA were hybridized for 16 hr at 45°. GeneChips were scanned using the Scanner 7G and the data was analyzed with Expression Console using Affymetrix default analysis settings and global scaling as normalization method. RMA analysis was employed to evaluate the gene expression. We used microarrays to detect the global gene expression in the carotid arteries of IRF4 knockout mice (IRF4 KO) compared with wild type mice (WT) at 14 days post-injury and identified distinct classes of altered genes
2019-10-15 | GSE88930 | GEO
Project description:Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle.
| S-EPMC4971827 | biostudies-literature
Project description:BACKGROUND AND AIMS:The uncertainty surrounding the role of Mycobacterium avium subsp paratuberculosis (Map) in Crohn's disease has been compounded by possible contamination from Map present in the lumen microflora. This study used laser capture microdissection (LCM) and polymerase chain reaction (PCR) to detect Map DNA in subepithelial granulomas, isolated from 15 surgically resected, formalin fixed specimens of granulomatous Crohn's disease and from 12 granulomatous disease controls (10 bowel, 2 non-bowel). METHODS:The effect of amplicon size on reliability of PCR from formalin fixed samples was examined by amplifying 435 bp and 133 bp sequences of the human APC gene. After this, nested primers were designed to detect a small fragment (155 bp) of the Map specific IS900 gene in Crohn's granulomas. LCM isolated granulomas from Map culture positive bovine intestine was used as positive control. PCR product specificity was confirmed by direct DNA sequencing. RESULTS:The smaller, but not the larger, fragment of the APC gene amplified reliably in all samples. Amplification of the 155 bp fragment of the IS900 gene detected Map DNA in microdissected Crohn's granulomas in 6 of 15 cases, and in 0 of 12 disease control granulomas. CONCLUSIONS:LCM can be used to detect Map DNA in granulomas in a proportion of patients with Crohn's disease. However, formalin fixation requires that comparatively short DNA fragments of the Map specific IS900 gene be targeted, to permit consistent detection. Detection of Map DNA within granulomas might suggest an infectious aetiology in a subset of patients; alternatively, a transmissible agent may not be involved but mycobacterial DNA may influence pathogenesis by modifying the local cytokine responses.
| S-EPMC1773423 | biostudies-literature
Project description:We evaluated the importance of tumor cell selection for generating gene signatures in non-small cell lung cancer. Tumor and nontumor tissue from macroscopically dissected (Macro) surgical specimens (31 pairs from 32 subjects) was homogenized, extracted, amplified, and hybridized to microarrays. Adjacent scout sections were histologically mapped; sets of approximately 1000 tumor cells and nontumor cells (alveolar or bronchial) were procured by laser capture microdissection (LCM). Within histological strata, LCM and Macro specimens exhibited approximately 67% to 80% nonoverlap in differentially expressed (DE) genes. In a representative subset, LCM uniquely identified 300 DE genes in tumor versus nontumor specimens, largely attributable to cell selection; 382 DE genes were common to Macro, Macro with preamplification, and LCM platforms. RT-qPCR validation in a 33-gene subset was confirmatory (ρ = 0.789 to 0.964, P = 0.0013 to 0.0028). Pathway analysis of LCM data suggested alterations in known cancer pathways (cell growth, death, movement, cycle, and signaling components), among others (eg, immune, inflammatory). A unique nine-gene LCM signature had higher tumor-nontumor discriminatory accuracy (100%) than the corresponding Macro signature (87%). Comparison with Cancer Genome Atlas data sets (based on homogenized Macro tissue) revealed both substantial overlap and important differences from LCM specimen results. Thus, cell selection via LCM enhances expression profiling precision, and confirms both known and under-appreciated lung cancer genes and pathways.
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Project description:Laser capture microdissection (LCM) allows the precise procurement of enriched cell populations from a heterogeneous tissue, or live cell culture, under direct microscopic visualization. Histologically enriched cell populations can be procured by harvesting cells of interest directly or isolating specific cells by ablating unwanted cells. The basic components of laser microdissection technology are (a) visualization of cells via light microscopy, (b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and (c) removal of cells of interest from the heterogeneous tissue section. The capture and cutting methods (instruments) for laser microdissection differ in the manner by which cells of interest are removed from the heterogeneous sample. Laser energy in the capture method is infrared (810 nm), while in the cutting mode the laser is ultraviolet (355 nm). Infrared lasers melt a thermolabile polymer that adheres to the cells of interest, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes LCM using an Arcturus(XT) instrument for downstream protein sample analysis and using an mmi CellCut Plus® instrument for RNA analysis via NanoString technology.
| S-EPMC3766583 | biostudies-literature
Project description:Different cell types and multiple cellular connections characterize the human brain. Gene expression analysis using a specific population of cells is more accurate than conducting analysis of the whole tissue homogenate, particularly in the context of neurodegenerative diseases, where a specific subset of cells is affected by the different pathology. Due to the difficulty of obtaining homogenous cell populations, gene expression in specific cell-types (neurons, astrocytes, etc.) has been understudied. To leverage the use of archive resources of frozen human brains in studies of neurodegenerative diseases, we developed and calibrated a method to quantify cell-type specific-neuronal, astrocytes-expression profiles of genes implicated in neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. Archive human frozen brain tissues were used to prepare slides for rapid immunostaining using cell-specific antibodies. The immunoreactive-cells were isolated by Laser Capture Microdissection (LCM). The enrichment for a particular cell-type of interest was validated in post-analysis stage by the expression of cell-specific markers. We optimized the technique to preserve the RNA integrity, so that the RNA was suitable for downstream expression analyses. Following RNA extraction, the expression levels were determined digitally using nCounter Single Cell Gene Expression assay (NanoString Technologies®). The results demonstrated that using our optimized technique we successfully isolated single neurons and astrocytes from human frozen brain tissues and obtained RNA of a good quality that was suitable for mRNA expression analysis. We present here new advancements compared to previous reported methods, which improve the method's feasibility and its applicability for a variety of downstream molecular analyses. Our new developed method can be implemented in genetic and functional genomic research of neurodegenerative diseases and has the potential to significantly advance the field.
| S-EPMC4988976 | biostudies-literature
Project description:The oxytocin (Oxt) and vasopressin (Avp) magnocellular neurons (MCNs) in the hypothalamus are the only neuronal phenotypes that are present in the supraoptic nucleus (SON), and are characterized by their robust and selective expression of either the Oxt or Avp genes. In this paper, we take advantage of the differential expression of these neuropeptide genes to identify and isolate these two individual phenotypes from the rat SON by laser capture microdissection (LCM), and to analyze the differential expression of several of their transcription factor mRNAs by qRT-PCR. We identify these neuronal phenotypes by stereotaxically injecting recombinant Adeno-Associated Viral (rAAV) vectors which contain cell-type specific Oxt or Avp promoters that drive expression of EGFP selectively in either the Oxt or Avp MCNs into the SON. The fluorescent MCNs are then dissected by LCM using a novel Cap Road Map protocol described in this paper, and the purified MCNs are extracted for their RNAs. qRT-PCR of these RNAs show that some transcription factors (RORA and c-jun) are differentially expressed in the Oxt and Avp MCNs.
| S-EPMC3722287 | biostudies-literature