Microarray expression profiling in the denervated hippocampus identifies long noncoding RNAs functionally involved in neurogenesis
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
PROVIDER: GSE96992 | GEO | 2017/03/28
SECONDARY ACCESSION(S): PRJNA380550
REPOSITORIES: GEO
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Project description:Purpose: Pulmonary tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has long been associated with the development of lung adenocarcinoma (LUAD) and sarcoidosis. These chronic lung diseases share histopathological similarities that challenge differential diagnosis. Methods: Human lung tissues were used for total RNA extraction with RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA purity was assessed using a Nanodrop ND-2000 (Thermo Scientific), and each RNA sample had an A260:A280 ratio above 1.8 and an A260:A230 ratio above 2.0. RNA integrity was evaluated using the Agilent 2200 TapeStation (Agilent Technologies), and each sample had the RNA Integrity Number (RIN) above 7.0. The ribosomal RNAs (rRNAs) were removed using Ribo-Zero rRNA Removal Kits (Illumina). RNA libraries were then constructed by NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) and evaluated using the Agilent 2200 TapeStation and Qubit® 2.0 (Life Technologies). Sequencing was performed by RiboBio Co., Ltd. on an Illumina HiSeq 3000 machine with paired-end 150-bp reads. The adaptors and low quality bases assessed using FASTQC 0.11.3 were trimmed by Trimmomatic 0.39 using the following options: TRAILING:20, MINLEN:235 and CROP:235. Trimmed reads were then aligned to the ensembl 79 (GRCh38.p2) reference genome using STAR 2.4.2a. FeatureCounts 1.6.2 was subsequently employed to convert aligned short reads into read counts for each sample. Genes with less than ten counts in two or more samples were removed. The data were then analyzed using R 3.4.4 and DEseq2 1.18.1. Differentially expressed genes of each disease group were identified using Wald statistics test, with fold-change > 2 and Benjamini–Hochberg (BH)-adjusted P < 0.1 as compared to NC group. Results: These findings provide novel pathogenic links and molecular markers for better understanding and differential diagnosis of pulmonary TB, LUAD and sarcoidosis.
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Project description:A large number of oral squamous cell carcinomas (OSCCs) are believed to be preceded by oral potentially malignant disorders (OPMD) that have an increased likelihood of malignant transformation compared to clinically normal mucosa. This study was performed to identify differentially expressed genes between OPMDs that underwent malignant transformation (MT) and those that did not, termed ‘non-transforming’ (NT) cases. Total RNA was extracted from formalin-fixed paraffin-embedded tissue biopsies of 20 OPMD cases with known clinical outcomes (10 MT vs. 10 NT). Samples were assessed for quantity, quality and integrity of RNA prior to sequencing. Analysis for differential gene expression between MT and NT was performed using statistical packages in R. Genes were considered to be significantly differentially expressed if the False Discovery Rate corrected p-value was < 0.05. RNA yield was variable but RNA purity was good (A260/A280 >1.90). Analysis of RNA-Sequencing outputs revealed 41 genes (34 protein-coding; 7 non-coding) that were significantly differentially expressed between MT and NT cases. The log2 fold change for the statistically significant differentially expressed genes ranged from -2.63 to 2.48, with 23 protein-coding genes being downregulated and 11 protein-coding genes being upregulated in MT cases compared to NT cases. Several candidate genes that may play a role in malignant transformation of OPMD have been identified. Experiments to validate these candidates are underway. It is anticipated that this work will contribute to better understanding of the aetiopathogenesis of OPMD and development of novel biomarkers.
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Project description:Fast and slow skeletal muscles show different characteristics and phenotypes. This data obtained from microarray includes the comparison of normal fast plantaris and slow soleus muscles of adult rats. Characters of slow muscle are strongly dependent on the level of muscular activity. Denervation silences the muscular activity. Therefore, we determined the effects of denervation on gene expression in slow soleus muscle of adult rats. Denervation was performed by transection (~5 mm) of left sciatic nerve at the gluteal level. No treatments were made in the normal control rats. Sampling of soleus and/or plantaris was performed in both normal and experimental groups 28 days after the surgery.
2014-10-04 | E-GEOD-62028 | biostudies-arrayexpress
Project description:Juvenile pilocytic astrocytoma (JPA) is one of the most common brain tumors in children. The expression profiles of 21 JPAs, determined using Affymetrix GeneChip U133A, were compared with subjects with normal cerebella. The genes involved in neurogenesis, cell adhesion, synaptic transmission, central nervous system development, potassium ion transport, protein dephosphorylation, and cell differentiation were found to be significantly deregulated in JPA. These 21 JPAs were further clustered into two major groups by unsupervised hierarchical clustering using a set of 848 genes with high covariance (0.5-10). Supervised analysis with Significance Analysis of Microarrays software between these two potential subgroups identified a list of significant differentially expressed genes involved in cell adhesion, regulation of cell growth, cell motility, nerve ensheathment, and angiogenesis. Immunostaining of myelin basic protein on paraffin sections derived from 18 incompletely resected JPAs suggests that JPA without myelin basic proteinâpositively stained tumor cells may have a higher tendency to progress. Experiment Overall Design: Under an Institutional Review Boardâapproved protocol, brain tumor tissues were obtained, after informed consent, from patients undergoing tumor resection or other tumor-related neurosurgical procedures at the Texas Children's Hospital, Baylor College of Medicine. Samples of normal cerebellar tissue (n = 2) were isolated from surgically removed tissue adjacent to resected tumor tissue. Portions of the tumors were fixed in 10% formaldehyde and embedded in paraffin for sectioning and pathologic reviews, and the residual tissue samples were snap-frozen in liquid nitrogen and stored at â80°C for RNA extraction. All samples were obtained at initial diagnosis with no prior exposure to chemotherapy or radiation therapy. Normal fetal brain RNA was obtained from Stratagene (La Jolla, CA) and a normal cerebellum RNA was from Ambion, Inc. (Austin, TX). Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) followed by DNase I treatment and clean-up on aRNeasy spin column (Qiagen, Valencia, CA). RNA quality and purity were assessed by agarose gel electrophoresis and absorbance measurement at A260/A280.
2008-10-26 | E-GEOD-12907 | biostudies-arrayexpress
Project description:This study was performed to evaluated RNA extraction and gene expression analysis of FFPE specimen stored for more than 20 years. Using long time stored FFPE material; large retrospective studies correlating molecular features with therapeutic response and clinical outcome, can be performed. Quantitative PCR (qPCR) was used to evaluate RNA extraction methods and to compare gene expression profiles of FFPE and fresh frozen (FF) tissue. Extracted RNA was subsequently subjected to microarray analysis and compared to qPCR data. The Ambion RecoverAll kit appears to be particularly suited for RNA extraction of long time stored FFPE tissues. Gene expression analysis using Affymetrix platform displayed a high degree of correlation for endogenous control genes comparing FF and FFPE tissues. We conclude that high quality gene expression signatures can be recognized using Affymetrix gene expression platform on FFPE tissue stored for more than 20 years. However, a general interpretation must be done with caution as different FFPE procedures have varying effects on RNA quality. Three RNA extraction methods from Roche (Basel, Switzerland), Ambion (Austin, TX, USA) and Qiagen (Hilden, Germany), designed for FFPE material was used. The methods were compared using qPCR. For the qPCR analysis, two different concentrations of input cDNA were used (20ng and 100ng) and 32 human endogenous control genes were examined. RNA from the Ambion FFPE kit was further analyzed by using microarray. Amplification of RNA prior to microarray analysis was performed using Nugen technologies (San Carlos, CA, USA). Nugen has developed amplification kits both for FFPE and FF materials: WT- ovation FFPE RNA amplification System V2 (Nugen FFPE), Ovation FF RNA Amplification System V2 (Nugen V2 FF) and Ovation FF Pico RNA Amplification System (Nugen PICO FF). The Nugen FFPE kit, designed for FFPE material was only used for FFPE tissue. The Nugen Pico FF kit, designed to target small amounts of FF RNA (>500 pg) and the Nugen V2 FF kit designed to target total FF RNA, were only used for FF tissue. Affymetrix standard amplification protocol (Affy FF) designed for FF RNA was also included as the standard method for amplification.
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Project description:Gastric cancer is the second most common cause of cancer-related death worldwide. Up to 80% of patients who undergo curative surgical resection develop locoregional or distant recurrence. A recent large-scaled meta-analysis on 3,838 patients from 17 trials has demonstrated survival benefit from adjuvant chemotherapy when compared to surgery alone. Furthermore, INT-0116 study has demonstrated a survival benefit from postoperative chemoradiation therapy with 5-fluorouracil and leucovorin in gastric cancer patients. Despite these advances, 5-year disease-free survival rates remain poor for patients diagnosed with stage III or IV gastric cancer (stage IIIA, 57.6%, stage IIIB, 39.6%; and stage IV 26.3%) underscoring the need for development of new targeted agents. On the other hand those diagnosed with stage Ib/II gastric cancer have moderate 5 year recurrence rate (76.2%) and suggest the presence of significant molecular heterogeneity with varying prognosis. We hypothesized that gene expression of profiling of formalin fixed paraffin embedded tumor (FFPET) samples using whole genome cDNA-mediated Annealing, Selection, Extension, and Ligation (WG-DASL) assay could be used to develop robust prognostic profiles for gastric cancer treated with chemoradiotherapy that are independent of clinicopathological features. We have identified and validated a gene expression signature that predicts recurrence after curative resection. We also performed pathway analyses to delineate aberrant pathways in aggressive gastric cancer which suggest targeted treatment strategies. RNA was extracted from 2-4 sections of 4-μm thick, FFPET sections. Non-tumor elements were removed by manual microdissection before transfer to the extraction tube guided by hematoxylin and eosin stained slides. The samples with RNA concentrations of <40 ng/μL, A260/A280 ratios <1.5 or A260/230 ratios <1.0 were considered as inadequate samples and were excluded from the analysis.
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