Transcriptomic analysis of midbrain and individual hindbrain rhombomeres in the chick embryo
ABSTRACT: A systematic survey of the transcriptional status of individual segments of the developing chick hindbrain (r1-5) and the adjacent region of the embryonic midbrain (m) during the HH11 stage of chick development Affymetrix Chicken GeneChip Expression Study Paralell comparison of defined regions of the neural tube during early chick development
Project description:Few families of signaling factors have been implicated in the control of development. Here we identify the neuropeptides nociceptin and somatostatin, a neurotransmitter and neuroendocrine hormone, as a class of developmental signals in chick and zebrafish. We show that signals from the anterior mesendoderm are required for the formation of anterior placode progenitors with one of the signals being somatostatin. Somatostatin controls ectodermal expression of nociceptin and both peptides regulate Pax6 in lens and olfactory progenitors. Consequently, loss of somatostatin and nociceptin signaling leads to severe reduction of lens formation. Our findings not only uncover these neuropeptides as developmental signals, but also identify a long-sought-after mechanism that initiates Pax6 in placode progenitors and may explain the ancient evolutionary origin of neuropeptides, pre-dating a complex nervous system. We used progenitors for anterior and posterior sensory placodes dissected from chick embryos HH5-7; these were either processed immediately or cultured for 5 hrs to hybridise to Affymetrix chick array. We aimed to identify genes that are co regualted with Pax6, a key regulator of lens and olfactory progenitor cells. Pax6 is normally present in anterior, but not posterior placode precursors, but upregulated in both after 5 hrs culture.
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.
Project description:In vertebrates the endoderm which gives rise to the epithelial lining of the digestive tract becomes regionalized along its antero-posterior axis after gastrulation. The molecular basis of the initial step of this regionalization has largely remained unclear. Using chick model, we generated high-quality transcriptomic datasets of different stages/regions of the endoderm and analyzed their molecular heterogeneity. Total RNA from HH stage 4-5 entire definitive endoderm, HH stages 10-11 foregut endoderm and HH stages 8-10 mid/hindgut endoderm were purified. 5 μg of RNA from each sample were used to screen Affymetrix Chicken Genome Array without an amplification step.
Project description:To further identify the transcriptional changes underlying congenital tracheal malformation in a1H knockout mice, the differential gene expression panel was examined by Affymetrix microarray. To investigate the roles of a1H-regulated genes in tracheal development, we characterize the unique transcriptional changes of early trachea in kockout mice, and compare the expression profiles of knock out trachea with those of wild type trachea. We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up or down-regulated genes during this process. Mouse embryos trachea were collected at embryo day 16 (E16) for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain homozygous mouse embryos trachea in order to compare with gene expression profile of wild type mouse tracheal.
Project description:Use existing public data, cell lines and patient tumors with a personalized medicine approach to predict effective therapies for treatment of Neurofibroma tumors. Compare expression profile to reference set to identify over expressed genes that are drugable targets.
Project description:To understand the underlying mechanism by which Alox15 gene is required by HSCs, we performed a comparative DNA microarray analysis using total RNA isolated from wild type Lin-Sca-1+c-Kit+, SELP-/- Lin-Sca-1+c-Kit+. The result was validated by quantitative real-time PCR analysis of wild type Lin-Sca-1+c-Kit+ and SELP-/- Lin-Sca-1+c-Kit+. Cancer stem cells are responsible for the initiation and maintenance of some types of cancer, and few effective target genes in these stem cells have been identified. Here we show that the selp is essential for the survival of leukemia stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML). To understand the underlying mechanism through which SELP regulates the function of HSCs, the gene expression profiles between WT and Selp-/- HSCs were compared. To understand the underlying mechanism through which SELP regulates the function of HSCs, the gene expression profiles between WT and Selp-/- HSCs were compared.
Project description:The purpose of the present study was to investigate the association of glutathione S-transferase P1 (GSTP1) expression with resistance to neoadjuvant paclitaxel followed by 5-fluorouracil/epirubicin/cyclophosphamide (P-FEC) in human breast cancers. The relationship of GSTP1 expression and GSTP1 promoter hypermethylation with intrinsic subtypes was also investigated. In this study, primary breast cancer patients (n = 123, stage II-III) treated with neoadjuvant P-FEC were analyzed. Tumor samples were obtained by vacuum-assisted core biopsy before P-FEC. GSTP1 expression was determined using immunohistochemistry, GSTP1 promoter methylation index (MI) using bisulfite methylation assay and intrinsic subtypes using DNA microarray. The pathological complete response (pCR) rate was significantly higher in GSTP1-negative tumors (80.0%) than GSTP1-positive tumors (30.6%) (P = 0.009) among estrogen receptor (ER)-negative tumors but not among ER-positive tumors (P = 0.267). Multivariate analysis showed that GSTP1 was the only predictive factor for pCR (P = 0.013) among ER-negative tumors. Luminal A, luminal B and HER2-enriched tumors showed a significantly lower GSTP1 positivity than basal-like tumors (P = 0.002, P < 0.001 and P = 0.009, respectively), while luminal A, luminal B and HER2-enriched tumors showed a higher GSTP1 MI than basal-like tumors (P = 0.076, P < 0.001 and P < 0.001, respectively). In conclusion, these results suggest the possibility that GSTP1 expression can predict pathological response to P-FEC in ER-negative tumors but not in ER-positive tumors. Additionally, GSTP1 promoter hypermethylation might be implicated more importantly in the pathogenesis of luminal A, luminal B and HER2-enriched tumors than basal-like tumors. Fresh frozen tumor samples obtained by vacuum-assisted core biopsy from one hundred and fifteen patients were subjected to RNA extraction and hybridization on Affymetrix microarrays.
Project description:Mutations in the genes encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types, resulting in production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). How mutant IDH and 2-HG alter signaling pathways to promote cancer, though, remains unclear. Additionally, there exist relatively few cell lines with IDH mutations. To examine the effect of endogenous IDH mutations and 2-HG, we created a panel of isogenic epithelial cell lines with either wild-type IDH1/2 or clinically relevant IDH1/2 mutations. Differences were noted in the ability of IDH mutations to cause robust 2-HG accumulation. IDH1/2 mutants that produce high levels of 2-HG cause an epithelial-mesenchymal transition (EMT)-like phenotype, characterized by changes in EMT-related gene expression and cellular morphology. 2-HG is sufficient to recapitulate aspects of this phenotype in the absence of an IDH mutation. In the cells types examined, mutant IDH-induced EMT is dependent on upregulation of the transcription factor ZEB1 and downregulation of the mir-200 family of microRNAs. Furthermore, sustained knockdown of IDH1 in IDH1 R132H mutant cells is sufficient to reverse many characteristics of EMT, demonstrating that continued expression of mutant IDH is required to maintain this phenotype. These results suggest mutant IDH proteins can reversibly deregulate discrete signaling pathways that contribute to tumorigenesis 9 HCT116 isogenic clones with wild-type or IDH1/2 mutations. Samples were analyzed in duplicate.
Project description:LNCaP-derived MDV3100-resistant clones were treated with MDV3100 for 24h prior to collection This experiment is designed to see if MDV3100 resistant clones remain responsive to MDV3100 Control and resistant clones were seeded in 6-well plates for 3d prior to treatment with DMSO or MDV3100 for 24h
Project description:MLL1 WT or KO MEF with and without HSP90 inhibitor treatment MEF cells were seeded in 6-well plates for 3d with 100ng/ml doxycycline prior to treatment with 0.1% DMSO or 100nM AUY922 for 3h