Comparison between conventionally-isolated and laser capture microdissected glomeruli
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
PROVIDER: GSE8998 | GEO | 2007/09/11
SECONDARY ACCESSION(S): PRJNA102473
REPOSITORIES: GEO
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Project description:We compared the gene expression analysis of 2 different glomerular isolation techniques (laser capture microdissection with 2 rounds of RNA amplification and unamplified glomerular RNA after iron perfusion with glomerular sieving) and obtained different results depending on the glomerular isolation technique that was used Experiment Overall Design: Paired kidneys from Lewis-Hsd rat kidneys were collected. The glomeruli of one kidney was obtained by LCM with 2 rounds of RNA amplification while the unamplified glomerular RNA of the contralateral kidney was obtained after perfusion with iron oxide and isolated by magnetic purification and a glomerular sieving technique. The RNA was analyzed using the Affymetrix Rat 230A microarrays.
2008-06-17 | E-GEOD-8998 | biostudies-arrayexpress
Project description:Alteration of fetal kidney morphogenesis in diabetic pregnancies is poorly described, especially changes in the extracellular matrix (ECM) and glomerular basement membrane (GBM) during glomerulogenesis. Addressing the ECM proteome, or matrisome, in mouse fetal glomeruli in a healthy and diabetic environment will improve understanding about the association between ECM and GBM changes in the glomerulus as potential reprogramming mechanisms for glomerular dysfunction in infants of mothers with diabetes. This project aimed to define the matrisome and BM composition in the maturing murine fetal glomeruli collected from normal and diabetic mothers. For this, we used laser microdissection microscopy to isolate maturing glomeruli from cryosections, and analyse by label-free tandem mass spectrometry to test the hypothesis that maternal diabetes influences ECM and GBM composition, assembly and function.
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Project description:Laser capture microdissection (LCM) is a superior method for nondestructive collection of specific cell populations from tissue sections. Although DNA, RNA, and protein have been analyzed from LCM-procured samples, epigenetic analyses, particularly of fetal, highly hydrated tissue, have not been attempted. A standardized protocol with quality assurance measures was established to procure cells by LCM of the medial edge epithelia (MEE) of the fetal palatal processes for isolation of intact microRNA for expression analyses and genomic DNA (gDNA) for CpG methylation analyses. MicroRNA preparations, obtained using the RNAqueous Micro kit (Life Technologies), exhibited better yields and higher quality than those obtained using the Arcturus PicoPure RNA Isolation kit (Life Technologies). The approach was validated using real-time polymerase chain reaction (PCR) to determine expression of selected microRNAs (miR-99a and miR-200b) and pyrosequencing to determine CpG methylation status of selected genes (Aph1a and Dkk4) in the MEE. These studies describe an optimized approach for employing LCM of epithelial cells from fresh frozen fetal tissue that enables quantitative analyses of microRNA expression levels and CpG methylation.
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Project description:The purpose of this study was to identify differentially expressed genes in laser-capture microdissected (LCM) invasive mammary carcinomas (IMCs). Invasive mammary carcinoma of the breast surgical resection specimens were laser capture microdissected for RNA extraction and hybridization to Affymetrix microarrays.
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Project description:The combination of stable isotope labeling of amino acids in mammals (SILAM) and laser capture microdissection (LCM) for selective proteomic analysis of the targeted tissues holds tremendous potential for refined characterization of proteome changes within complex tissues such as the brain. The authors have applied this approach to measure changes in relative protein abundance in ventral tegmental area (VTA) of the rat brain that correlate to pharmacological perturbations. Enriched (13)C6(15)N2-lysine was introduced in vivo via diet. These animals were sacrificed during the middle of the 12-hour light period to extract isotopically "heavy" proteins, which were then used as a reference for extracts from dosed, unlabeled rats. Animals were administered an orexin peptide (Ox-B), an orexin receptor antagonist (ORA), or a mixture of both (Ox-B + ORA). All samples were obtained at same phase of the sleep cycle. Labeled-pair identification and differential quantitation provided protein identification and expression ratio data. Five proteins were found to exhibit decreased relative abundance after administration of an ORA, including α-synuclein and rat myelin basic protein. Conversely, six proteins showed increased relative abundance upon antagonist treatment, including 2',3'-cyclic nucleotide 3'-phosphodiesterase.
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Project description:BACKGROUND:IgA nephropathy (IgAN) involves mesangial matrix expansion, but the proteomic composition of this matrix is unknown. The present study aimed to characterize changes in extracellular matrix in IgAN. METHODS:In the present study we used mass spectrometry-based proteomics in order to quantitatively compare protein abundance between glomeruli of patients with IgAN (n?=?25) and controls with normal biopsy findings (n?=?15). RESULTS:Using a previously published paper by Lennon et al. and cross-referencing with the Matrisome database we identified 179 extracellular matrix proteins. In the comparison between IgAN and controls, IgAN glomeruli showed significantly higher abundance of extracellular matrix structural proteins (e.g periostin, vitronectin, and extracellular matrix protein 1) and extracellular matrix associated proteins (e.g. azurocidin, myeloperoxidase, neutrophil elastase, matrix metalloproteinase-9 and matrix metalloproteinase 2). Periostin (fold change 3.3) and azurocidin (3.0) had the strongest fold change between IgAN and controls; periostin was also higher in IgAN patients who progressed to ESRD as compared to patients who did not. CONCLUSION:IgAN is associated with widespread changes of the glomerular extracellular matrix proteome. Proteins important in glomerular sclerosis or inflammation seem to be most strongly increased and periostin might be an important marker of glomerular damage in IgAN.
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Project description:Abnormal extracellular matrix (ECM) remodeling is a prominent feature of many glomerular diseases and is a final common pathway of glomerular injury. However, changes in ECM composition accompanying disease-related remodeling are unknown. The physical properties of ECM create challenges for characterization of composition using standard protein extraction techniques, as the insoluble components of ECM are frequently discarded and many ECM proteins are in low abundance compared to other cell proteins. Prior proteomic studies defining normal ECM composition used a large number of glomeruli isolated from human kidneys retrieved for transplantation or by nephrectomy for cancer. Here we examined the ability to identify ECM proteins by mass spectrometry using glomerular sections compatible with those available from standard renal biopsy specimens. Proteins were classified as ECM by comparison to the Matrisome database and previously identified glomerular ECM proteins. Optimal ECM protein identification resulted from sequential decellularization and protein extraction of 100 human glomerular sections isolated by laser capture microdissection from either frozen or formalin-fixed, paraffin-embedded tissue. In total, 147 ECM proteins were identified, including the majority of structural and GBM proteins previously identified along with a number of matrix and glomerular basement membrane proteins not previously associated with glomeruli. Thus, our study demonstrates the feasibility of proteomic analysis of glomerular ECM from retrieved glomerular sections isolated from renal biopsy tissue and expands the list of known ECM proteins in glomeruli.
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Project description:The specific contribution of the two TNF-receptors Tnfr1 and Tnfr2 to TNF-induced inflammation in the glomerulus is unknown. In mice, TNF exposure induces glomerular expression of inflammatory mediators like adhesion molecules and chemokines in vivo, and glomerular accumulation of leukocytes. To examine Tnfr-specific inflammatory responses in intrinsic glomerular cells but not infiltrating leukocytes we performed microarray gene expression profiling on intact glomeruli isolated from wild-type and Tnfr-deficient mice following TNF exposure in vitro. Glomeruli were isolated from male C57BL/6J wild-type and Tnfr-deficient mice applying a magnetic bead-based isolation technique. Isolated intact glomeruli were stimulated with TNF for 12 hours in vitro for subsequent RNA extraction and hybridization on Affymetrix microarrays.
2013-09-11 | E-GEOD-43928 | biostudies-arrayexpress
Project description:Mass spectrometry (MS)-based label-free proteomics offers an unbiased approach to screen biomarkers related to disease progression and therapy-resistance of breast cancer on the global scale. However, multi-step sample preparation can introduce large variation in generated data, while inappropriate statistical methods will lead to false positive hits. All these issues have hampered the identification of reliable protein markers. A workflow, which integrates reproducible and robust sample preparation and data handling methods, is highly desirable in clinical proteomics investigations. Here we describe a label-free tissue proteomics pipeline, which encompasses laser capture microdissection (LCM) followed by nanoscale liquid chromatography and high resolution MS. This pipeline routinely identifies on average ?10,000 peptides corresponding to ?1,800 proteins from sub-microgram amounts of protein extracted from ?4,000 LCM breast cancer epithelial cells. Highly reproducible abundance data were generated from different technical and biological replicates. As a proof-of-principle, comparative proteome analysis was performed on estrogen receptor ? positive or negative (ER+/-) samples, and commonly known differentially expressed proteins related to ER expression in breast cancer were identified. Therefore, we show that our tissue proteomics pipeline is robust and applicable for the identification of breast cancer specific protein markers.
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Project description:The Wilms tumor-suppressor gene WT1, a key player in renal development, also has a crucial role in maintenance of the glomerulus in the mature kidney. However, molecular pathways orchestrated by WT1 in podocytes, where it is highly expressed, remain unknown. Their defects are thought to modify the cross-talk between podocytes and other glomerular cells and ultimately lead to glomerular sclerosis, as observed in diffuse mesangial sclerosis (DMS) a nephropathy associated with WT1 mutations. To identify podocyte WT1 targets, we generated a novel DMS mouse line, performed gene expression profiling in isolated glomeruli, and identified excellent candidates that may modify podocyte differentiation and growth factor signalling in glomeruli. Scel, encoding sciellin, a protein of the cornified envelope in the skin, and sulf1, encoding a 6-O endosulfatase, are shown to be expressed in wild type podocytes and to be strongly down-regulated in mutants. Co-expression of Wt1, Scel and Sulf1 was also found in a mesonephric cell line, and siRNA-mediated knockdown of WT1 decreased Scel and Sulf1 mRNAs and proteins. By ChIP we show that Scel and Sulf1 are direct WT1 targets. Cyp26a1, encoding an enzyme involved in the degradation of retinoic acid, is shown to be up-regulated in mutant podocytes. Cyp26a1 may play a role in the development of glomerular lesions but does not seem to be regulated by WT1. These results provide novel clues in our understanding of normal glomerular function and early events involved in glomerulosclerosis. Experiment Overall Design: Isolation of glomeruli from mutant (FVB-N4 Wt1+/R394W) and wild-type (FVB-N4 Wt1+/+) was performed after cardiac Dynabead perfusion. GeneChip analysis of glomeruli from 5 Wt1+/R394W mice and 5 Wt1+/+ littermates (N4-FVB) were performed independently. Animals were unweaned 27-day-old males. The Wt1+/R394W mice used were showing little albuminuria (<3 ug/ul on Coomassie blue stained SDS-PAGE gel) and no evidence of mesangial lesions by light microscopy.
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