Ena-DATASET-NCCS-29-08-2019-09:06:17:965-16 - samples
Ontology highlight
ABSTRACT:
PROVIDER: EGAD00001005303 | EGA |
REPOSITORIES: EGA
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Project description:Whole-genome sequencing (WGS) was performed for 13 pairs of tumor-normal samples from patients diagnosed with NKTL. Genomic DNA from tumor tissue was extracted with QIAamp DNA Mini Kit. The DNA for the matching normal was obtained from blood or buccal swabs and purified by Blood and Cell Culture DNA Mini kit or E.Z.N.A. Tissue DNA Kit (Omega Bio-tek) according to manufacturer’s instructions. The quantity and quality were assessed by Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) and agarose gel electrophoresis. All sequencing libraries were prepared using TruSeq Nano DNA Library Prep Kit (Illumina). Paired-end sequencing was performed on Illumina HiSeq 2000 or HiSeq X Ten as 2x101 bp or 2x151 bp, respectively.
8 NKTL FFPE specimens were screened for somatic mutations using deep targeted capture sequencing (TCS). FFPE rolls or slides were extracted using QIAamp DNA FFPE Tissue kit (QIAGEN). The FFPE genomic DNA was treated with NEBNext FFPE DNA Repair Mix and assessed by Quant-it PicoGreen dsDNA Assay Kit (Invitrogen). The library was generated from 10-200 ng DNA with SureSelectXT Low Input Target Enrichment System for Illumina Paired-End Sequencing Library (Agilent Technologies) according to manufacturer’s instructions. RNA based probe was designed with SureDesign (Agilent Technologies) to target-capture 140 genes. Next, the captured libraries were pooled in equimolar concentration and sequenced on Illumina Novaseq 6000 platform with SP or S1 chip. Reads aligning to 40 selected genes were isolated post-alignment for this submission.
Prefix used in filenames:
T - Tumor samples
N - Matched-Normal samples
| EGAD00001004140 | EGA
Project description:We performed a comprehensive genome-wide miRNA expression profiling (MEP) of extranodal nasal-type Natural Killer/T-cell lymphoma (NKTL) using formalin fixed paraffin-embedded tissue (FFPE) (n=30) and NK cell lines (n=6) in comparison with normal NK cells, with the objective of understanding the pathogenetic role of miRNA deregulation in NKTL. Total RNA, including miRNA, were extracted using Ambion Recoverall Kit and profiled using Agilent human miRNA V2.
2011-11-23 | E-GEOD-31377 | biostudies-arrayexpress
Project description:Human PDCD4 wild-type (wt) promoter fragments were amplified from U2OS genomic DNA and X-box deletion mutants (mut) were generated using the QuikChange site-directed mutagenesis kit (Agilent Technologies). DNA probes for affinity purification with the same sequences were obtained by PCR using a biotinylated primer for labeling the 3' end (Thermo Fisher Scientific). DNA affinity purification with nuclear extracts from Nutlin-3a treated U2OS cells.
2024-08-08 | PXD020932 | Pride
Project description:To investigate the effect of CodY mutation on the gene expression in Streptococcus suis serotype 2 SC19 strain, we have employed whole genome microarray expression profiling as a discovery platform to identify genes regulated by CodY mutation. DNA microarray analysis was performed using an Agilent custom-designed oligonucleotide microarray. Based upon the whole genome sequence of SC19 , specific 60-mer oligonucleotide probes were designed using eArray (https://earray.chem.agilent.com/earray/), to cover all annotated genes. Probes were printed seven times on microarray slides. Three biological replicates of total RNA from two wild type strains and from two codY mutant strains were amplified and labeled with Cy3-CTP using Low Input Quick Amp Labeling Kit, one-color(Agilent technologies, US), following the manufacturer’s instructions. Labeled cRNA was purified using the RNeasy mini kit (Qiagen). After fragmentation, microarray slides were hybridized with 600 ng Cy3-labeled cRNA. Hybridization was performed at 65 °C for 17 h with rotation at 10 rpm. Microarray slides were washed and scanned by an Agilent Microarray Scanner (G2565BA). Those genes with greater than two-fold change ratios were regarded as differentially expressed genes.
2015-04-21 | GSE68068 | GEO
Project description:MicroRNAs (miRNAs) are known to be deregulated in human breast cancer (BC). The purpose of the current study was to investigate the expression of miRNAs in different stages of BC to assess their biological value in BC progression. MiRNA expression was assessed in a series of BC patients (n=7) with distinct stages of tumour progression (Normal, in-situ (DCIS), primary invasive BC and nodal metastases) to evaluate miRNA differential expression. We used an Agilent miRNA microarray based platform which uses miRBase 16 to screen for 1205 Homo sapiens (hsa) and 144 human viral miRNA candidates. To validate the microarray data, the expression of two deregulated miRNAs was measured by TaqMan quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). This study was conducted Formalin Fixed and Paraffin Embedded (FFPE) specimens from 7 female patients. Twenty M-NM-<m thick FFPE sections were cut and mounted on PALM Membrane Slides. Under direct microscopic visualization, tissues of interest were micro-dissected using PALM non-contact Laser catapulsion instrument (P.A.L.M. Microlaser Technologies, Carl Zeiss Ltd). Total RNA, including miRNAs, was extracted with the TRIzol reagent (Invitrogen, supplied by Fisher Scientific UK Ltd) according to the manufacturerM-bM-^@M-^Ys instructions. Total RNA was quantified with Quant-iT RiboGreen RNA Quantitation Kit (Fisher Scientific UK Ltd), which is an accurate method of measuring total RNA with a detection limit of 0.001-1ng/M-BM-5l. RNA purity and RNA integrity number (RIN) were determined on an Agilent 2100 Bioanalyzer and RNA 6000 NanoLabChip Kits (both Agilent Technologies, Santa Clara, USA), and the RIN of all the samples was >2 but <3. This is expected for FFPE breast samples and is acceptable by Agilent Technologies, Santa Clara, USA, for FFPE samples to undergo miRNA microarray analysis. MiRNAs extracted from laser microdissected tissue components were profiled using Agilent microRNA microarray profiling system (Agilent Technologies, Santa Clara, USA). Total RNA samples were spiked using the MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA) to evaluate efficiencies of labelling and hybridisation. Total RNA was treated with Calf Intestinal Alkaline Phosphatase (CIP), and then 100 ng of total RNA was used per sample to initiate a labelling reaction. Ligation master mix for T4 RNA ligase, which includes Cyanine 3-Cytidine biphosphate (Cyanine 3-pCp) (Complete miRNA Labelling and Hyb Kit, Agilent Technologies, Santa Clara, USA) was used to label the dephosphorylated RNA. Cyanine-3-labelled miRNA samples were hybridised to human miRNA microarrays (Release 16.0, 8x60K) (Agilent Technologies, Santa Clara, USA) at 55M-BM-0C for 20 hrs. Microarray slides were washed with increasing stringency (Gene Expression Wash Buffers, Agilent Technologies, Santa Clara), and subsequently dried with acetonitrile (Sigma-Aldrich, St. Louis, USA). Fluorescent signal intensities were detected on an Agilent Microarray Scanner (Agilent Technologies, Santa Clara, USA) using the Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA) and extracted from the images with the Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA).
2012-06-22 | E-GEOD-38867 | biostudies-arrayexpress
Project description:Total RNA was isolated from Peripheral blood (PB) and Bone Marrow (BM) samples using mirVana kit and RNA integrity number (RIN) was determined in Agilent Bioanalyzer (2100) to assess the suitability for microarray assays. Fifty samples were individually analyzed using miRCURY™ LNA arrays version 11.0 (Exiqon, Denmark). The LNA array slides were scanned using Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and image analysis was carried out using ImaGene 8.0 software (BioDiscovery, Inc., USA). The quantified signals were background corrected (Normexp with offset value 10 14 and normalized using global Lowess (Locally Weighted Scatterplot Smoothing) regression algorithm.
2014-07-09 | GSE59199 | GEO
Project description:Total RNA was isolated from epithelial tissues of different grades and stages of human breast cancer samples using mirVana kit and RNA integrity number (RIN) was determined in Agilent Bioanalyzer (2100) to assess the suitability for microarray assays. Sixteen samples were individually analyzed using miRCURY™ LNA arrays version 11.0 (Exiqon, Denmark). The LNA array slides were scanned using Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and image analysis was carried out using ImaGene 8.0 software (BioDiscovery, Inc., USA). The quantified signals were background corrected (Normexp with offset value 10 14 and normalized using global Lowess (Locally Weighted Scatterplot Smoothing) regression algorithm.
2014-08-26 | GSE60725 | GEO
Project description:Light-controlled in situ synthesis of DNA microarrays (Affymetrix GeneChip® Mouse Genome 430 2.0) were performed to determine the altered gene expression. Affymetrix algorithm and multiple analysis comparison software were used for assessing gene expression differences, and mRNAs that increased or decreased in the brains of NP(α-M)-treated mice relative to that in the brains of Blank-NP-treated mice were identified. For the assay, total RNA was extracted using Takara RNAiso Plus Kit (Cat#9109, Takara, DaLian, LiaoNing, CN) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US). Qualified total RNA was further purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany). Array hybridization and wash was performed using GeneChip® Hybridization, Wash and Stain Kit (Cat#900720, Affymetrix, Santa Clara, CA, US) in Hybridization Oven 645 (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, US) and Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, US) followed the manufacturer’s instructions. Slides were scanned by GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 4.0 (Affymetrix, Santa Clara, CA, US) with default settings. Raw data were normalized by MAS 5.0 algorithm, Gene Spring Software12.6.1 (Agilent technologies, Santa Clara, CA, US).
2018-09-14 | GSE119904 | GEO
Project description:We used BAF250b-/- ES cells, two independently derived clones, at 18 and 72 hr in culture and compared them with parental cell line (wild-type) at the same time in culture (two replications each). Total RNAs were extracted using Trizol (Invitrogen) according to manufacturer protocol. 2.5 ug of total RNA samples were labeled with Cy3-CTP using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent). A reference target (Cy5-CTP-labeled) was prepared from the Universal Mouse Reference RNA (Stratagene). Labeled targets were purified using an RNeasy Mini Kit (Qiagen) according to the Agilent's protocol, quantitated by a NanoDrop scanning spectrophotometer (NanoDrop Technologies), and hybridized to the NIA Mouse 44K Microarray v2.2 (whole genome 60-mer oligo; manufactured by Agilent Technologies, #014117) (Carter et al. 2005) according to the Agilent protocol (G4140-90030; Agilent 60-mer oligo microarray processing protocol - SSC Wash, v1.0). All hybridizations were carried out in the two color protocol by combining one Cy3-CTP-labeled experimental target and Cy5-CTP-labeled reference target. Microarrays were scanned on an Agilent DNA Microarray Scanner, using standard settings, including automatic PMT adjustment. Keywords: genetic modification design,time series design
2008-06-09 | GSE8996 | GEO
Project description:DNA methylation arrays for 8 Surgical specimens(DNA extracted from FFPE slides) after cetuximab treatment
2020-01-17 | GSE110995 | GEO