Project description:To investigate the cytogenetic and large-scale chromosomal changes in involuted or non-involuted microGISTs using post-whole genome amplification (WGA) FFPE DNA materials Sixteen patients, total 19 FFPE tumor samples (block storage time 4 months to 9 years), including 16 microGISTs and 3 GISTs larger than 1 cm from the same patients harboring microGISTs. All FFPE tumor samples underwent DNA extraction and WGA (modified degenerate oligonucleotide PCR (DOP) method, provided by Sigma). For each tumor sample, a post-WGA DNA extract from the normal tissue in the same block (or block from the same patient with a block storage time differences less than 2 years) was obtained for tumor sample DNA co-hybridization. Tumor and normal areas of interest were marked and collected from 5- to 10-micron unstained or hematoxylin-stained sections by manual or laser (PixCell IITM, Arcturus Bioscience, CA, USA) microdissection. DNAs were then extracted. WGA was performed using GenomePlex® Tissue Whole Genome Amplification WGA5 kit (Sigma, Saint Louis, MO, USA; http://www.sigmaaldrich.com/) in parallel in accordance with the manufacturer's protocols. At least four independent experiments were concurrently performed per template amplification. Four separate WGA reaction products were pooled for each sample.
Project description:Soft tissue sarcomas (STS) often present a significant diagnostic challenge as many STS bear histologic resemblance, but are known to have very different clinical and biologic characteristics. Some STS subtypes are characterized by specific genetic abnormalities and this has helped in their classification, diagnosis and even treatment. However, a large majority of STS have no known specific genetic aberrations even though they almost always have highly aberrant karyotypes. We therefore hypothesize that the latter subgroup of STS bear genetic abnormalities that are sub-type specific, but as yet unidentified. High-resolution mapping of copy number aberrations in cancer genomes is a valuable way of identifying recurrent genomic changes that could be of pathogenetic significance. Traditionally, this has been done using high quality DNA obtained from fresh frozen tissue or cells and archived tissue is generally regarded as unsuitable because of the degradative effects of formalin fixation on DNA. Utility of archival tumour material for such molecular genetic analysis is vital, especially for rare cancers like STS but recent efforts to accomplish this have produced variable results. We therefore set out, in addition to optimize a protocol for obtaining genomic copy number data from formalin-fixed, paraffin-embedded (FFPE) STS material that is comparable to that from fresh frozen (FF) material. Microarray-based Comparative Genomic Hybridization (aCGH), a high- resolution, genome-wide method was used to identify somatic copy number aberrations (SCNAs) in primary STS samples (fresh frozen and archival FFPE), using an optimized protocol for labeling DNA. Findings were confirmed using Conventional Cytogenetics and Fluorescence in-situ Hybridization (FISH). Data obtained from paired samples (FF and FFPE) of the same tumours showed similar results and array results were consistently of good quality. On-going analysis of the recurrent SCNAs in combination with expression data and clinical correlates may serve to identify specific patterns that can serve as diagnostic markers, characterize subgroups with prognostic implication or identify potential therapeutic targets. To identify common CNAs among LMS fresh and FFPE. 25 samples in total: 22 individual FFPE cases; 3 cases also obtained fresh. Reference DNA was obtained from same patient when possible, otherwise commercial genomic DNA was used [Promega® UK with Cat Nos. G1471 (male) and G1521 (female)].
Project description:Genetic variations play an important role in tumor development and metastasis. Hepatocellular carcinoma (HCC) is one of leading cause of cancer-related death. Despite improvements in surveillance and clinical treatment strategies, the prognosis of HCC remains dismal. Affymetrix SNP 6.0 array were used to evaluate the genetic characteristics of tumor DNA in 30 HBV-related HCC patients who were underwent liver transplantation. Recurrence related SNPs were selected and validated. Affymetrix SNP 6.0 arrays were performed according to the manufacturer's directions on DNA extracted from formalin-fixed paraffin-embedded HCC tissues
Project description:Distinct shaping of the upper versus lower facial skeleton is essential for function of the vertebrate jaw and middle ear, yet the cellular mechanisms by which this occurs have remained unclear. Here, we show that Endothelin1 (Edn1) signaling accelerates mesenchymal condensation and subsequent cartilage formation in the lower face through antagonism of Jagged-Notch signaling and Prrx1 transcription factors. A genomic analysis of facial skeletal precursors in mutants and overexpression embryos reveals that Jagged-Notch signaling represses genes that are strongly induced as pharyngeal arch neural crest-derived cells begin skeletal differentiation. In wild types, initial Jagged-Notch repression dorsally ensures that barx1+ condensations and cartilage differentiation occur first in ventral-intermediate zones of the pharyngeal arches. Reduced Jagged-Notch signaling results in an expansion of pre-cartilage condensations in the upper face, with loss of barx1 partially restoring dorsal cartilage shapes in jag1b mutants. Further, by studying new mutants for zebrafish prrx1a and prrx1b, we find that Prrx1 genes function in parallel to Jagged-Notch signaling to restrict the formation of dorsal barx1+ pre-cartilage condensations. Consistently, combined losses of jag1b and prrx1a/b robustly rescue ventral barx1+ condensations and lower facial cartilage development in edn1 mutants. Together, our work suggests that Edn1 works through parallel inhibition of Jagged-Notch and Prrx1 pathways to promote an earlier and more extensive establishment of cartilage condensations in the lower face. We performed RNAseq on FACS-sorted neural crest-derived pharyngeal arch cells (fli1a:GFP; sox10:DsRed double positive) from wild-type embryos at 3 different stages (20, 28, and 36 hours post fertilization) and embryos with altered levels of Edn1 and Notch signaling (edn1 mutants and hsp70I:Gal4; UAS:Edn1 transgenics; jag1b mutants, dibenzazepine-treated embryos, and hsp70I:Gal4; UAS:NICD transgenics. We also sequenced RNA from heat-shocked UAS:Edn1+ and hsp70I:Gal4+ transgenics and jag1b+/+ controls.
Project description:We obtained gene-expression profiles of microdissected renal-tubule cells from patients with proteinuric nephropathies. Based on the renal function during follow-up, the patients were divided in stable (n=14) and progressive (n=7) subjects (table 1). The cells of interest were laser-capture microdissected from frozen sections from archived kidney biopsy material. Initially all samples were processed as technical duplicates (2 x 21 arrays); due to a large number of signal-negative spots several arrays were excluded leaving 36 arrays for analysis. The samples P2, P6, P7, S10, S13 and S14 were analysed as individual arrays, all other samples were analysed after combination of duplicate arrays. Quality control: To test for reproducibility we calculated the intra-array variability of the duplicate arrays. Duplicate arrays were combined before statistical analysis where applicable. Patient and control characteristics can be found in the manuscript and on our website Frozen kidney biopsies were stained for alkaline phospatase, then the tubule cells were lasercapture microdissected using the PixCell II Laser Capture Microdissection System and CapSure; LCM Caps. Set of arrays that are part of repeated experiments Disease State: samples from patients with proteinuric nephropathy (stable or progressive) Biological Replicate Computed
Project description:Biologic agents active in other autoimmune settings have had variable effectiveness in newly diagnosed type 1 diabetes (T1D) where treatment across therapeutic targets is accompanied by transient stabilization of C-peptide levels in some patients, followed by progression at the same rate as in control groups. Why disparate treatments lead to similar clinical courses is currently unknown. Here, we use integrated systems biology and flow cytometry approaches to elucidate immunologic mechanisms associated with C-peptide stabilization in T1D subjects treated with the anti-CD3 monoclonal antibody, teplizumab. This work is part of the Immune Tolerance Network AbATE study (Autoimmunity-Blocking Antibody for Tolerance in Recently Diagnosed Type 1 Diabetes); data are also available through the ITN TrialShare portal: https://www.itntrialshare.org/project/Studies/ITN027AIDB/Study%20Data/begin.view?. We performed bulk RNA-seq on 63 sorted CD8+ T-cell samples from 3 responders at visit month 6 (N = 34, 15, 14); we sorted cells with and without EOMES-associated inhibitory receptors (TIGIT, KLRG1); prior to RNA extraction, cells were either unstimulated, stimulated with anti-CD3 and anti-CD28, or stimulated with additional PVR-Fc treatment.