NimbleGen Human DNA Methylation 3×720 K CpG Island Plus RefSeq Promoter Array (Roche) based on the HG18 genome release was used. The array contained 720,000 probes of 50–75 bp in length with a median probe spacing of 104 bp, covering 30,848 transcripts, 22,532 promoters, and 27,728 CpG islands. The raw intensities of the scanned image for both Cy5 and Cy3 channels were extracted using the NimbleScan (Roche). The raw intensity pair files were imported into the R statistical programming environment using custom R software.
Nimblescan
Manufactured by Roche
Sourced in United States
NimbleScan is a laboratory equipment product designed for efficient data acquisition and analysis. It provides a reliable platform for researchers to capture and process information from various experimental setups. The core function of NimbleScan is to facilitate the accurate collection and management of data, supporting scientific investigations and research endeavors.
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Lab products found in correlation
Genepix 4000b scanner, by Molecular Devices (3 mentions)
Axon genepix 4000b, by Molecular Devices (2 mentions)
Signalmap, by Roche (2 mentions)
Elx800 absorbance microplate reader, by Agilent Technologies (1 mentions)
Agilent genespring software, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Nimblegen hybridization system, by Roche (1 mentions)
Nimblegen one color dna labeling kit, by Roche (1 mentions)
Genepix pro software, by Molecular Devices (1 mentions)
Dneasy tissue kit, by Qiagen (1 mentions)
Genespring gx, by Agilent Technologies (1 mentions)
Nexus 6, by BioDiscovery (1 mentions)
Gentra puregene tissue kit, by Qiagen (1 mentions)
Human genomic dna, by Promega (1 mentions)
Nimblegen ms 200 microarray scanner, by Roche (1 mentions)
Nimblegen dual colordna labeling kit, by Roche (1 mentions)
16 protocols using nimblescan
1
Comprehensive DNA Methylation Profiling
2
Genome-wide DNA Methylation Analysis
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Raw data extraction to generate pair files employed NimbleScan (Roche NimbleGen, Pleasanton, CA, USA). Median-centering quantile normalization and linear smoothing were performed utilizing the Ringo, limma, and MEDME packages. Then, a normalized log2-ratio value (∗_ratio.gff file) was generated for every sample, from which enriched peaks (sliding window width, 1500 bp; mini probes/peak, 2; P value minimum cutoff (–log10), 2; maximum length between neighboring probes in a peak, 500 bp) were identified with NimbleScan 2.5 (Roche NimbleGen). Upon obtaining ∗_peaks.gff files, the determined peaks were mapped to transcripts and CpG islands. Differentially enriched peaks (DEPs) were determined by the M’ method, which reveals substantial gene changes. Next, log2 ratios for all probes in the DEP region were utilized to carry out a cluster analysis to directly reveal the methylation of DEP probes in various samples. Genes with DEPs in their promoters were considered differentially methylated genes (DMGs) and underwent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses. The promoters were classified into high (HCP), intermediate (ICP), and low (LCP) CpG promoters/regions, with high, intermediate, and low CpG density promoters, respectively.
3
RNA Isolation and Microarray Analysis of IUGR Placenta
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Hybridizations RNA isolation of 12 control and 12 IUGR placenta samples was performed according to standard protocols using Trizol reagent and samples were quantitatively and qualitatively controlled (37) . Three individual placenta samples within a group were then pooled, to produce four pools of vascular IUGR and four pools of normal pregnancy for the control group. The eight pools were then evaluated for purity, integrity, and quantification by the Agilent Bioanalyser 2100 R . One microgram total RNA from each pool was reverse transcribed, labeled, and then hybridized to NimbleGen Human HG18 60mer 4x72K arrays representing 23,456 human transcripts. Image analysis was performed with the NimbleScan software (Roche NimbleGen, Madison, WI), and feature intensities were exported as pair files. ArrayStar 3.0 R software (DNASTAR, Madison, WI) was used for probe summarization and normalization (RMA algorithm, quantile normalization), statistical analysis of differentially expressed genes (Student's t-test with Benjamini-Hochberg false discovery rate correction), and gene ontology analysis. The entire microarray data set is available at the EMBL-EBI ArrayExpress site under the reference E-MTAB-1956.
4
Genome-wide Gene Expression Profiling
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Total RNA was extracted using either TRIzol reagent (Invitrogen) or an RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturers' protocols. Following quality confirmation, RNA samples were amplified and labeled using a NimbleGen One Color DNA Labeling kit (Roche, Madison, WI, USA). RNA was then hybridized (NimbleGen Hybridization System, Roche, Madison, WI, USA) to a human genome array (12 × 135 K array; Roche) containing oligos representing 45,033 human genes. After washing, the processed slides were scanned with an ELx800 absorbance microplate reader (Biotek Instruments Inc., Winooski, VA, USA). NimbleScan (version 2.6., Roche NimbleGen, Madison, WI, USA) was used for data analysis, including quantile normalization and background correction. Agilent GeneSpring software (Agilent Technologies, Santa Clara, CA, USA) was used to further analyze the gene summary files. Three samples were randomly selected from each group for microarray analysis. The genes were considered differentially expressed when the ratio was >1.5. or <0.6.7 in normalized intensity between the two groups or when P-values were less than 0.0.5 by one-way analysis of variance (ANOVA).
5
Genomic DNA Profiling via Microarray Analysis
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Genomic DNA was extracted using the DNeasy Tissue Kit as per manufacturer's instructions (Qiagen, Valencia, CA). Pooled female genomic DNA (Promega, Madison, WI) was differentially labeled as a control sample. Hybridization was performed on 135,000 oligonucleotide arrays (Roche-Nimblegen, Madison, WI) with an average probe spacing every 35 kb according to manufacturer's recommendations. Arrays were imaged using a Genepix 4000B Scanner with GenePixPro software (Molecular Devices, Sunnyvale, CA), and data was analyzed with the NimbleScan and Genoglyphix software (Roche-Nimblegen). Variants were defined as segments that have at least 5 probes and a minimum log ratio of 0.3, 200 probes and a log ratio of 0.2 or 500 probes and a log ratio of 0.1. Variants were compared against public databases, including the Database of Genomic Variants (The Hospital for Sick Children, Toronto, Canada) and the Copy Number Variation Project at the Children's Hospital of Philadelphia to benign population variants. Polyclonal populations of naïve and CDKi-resistant cells were analyzed.
6
Potato Microarray Transcriptome Analysis
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We used 56 K potato microarray to perform a total of nine experiments (without replication) that compared gene expression among the two mutant lines and the control cultivar at three time points. The potato 56 K microarray was manufactured by Roche NimbleGen.20 The microarray was designed from 43,553 coding sequences of PGSC version 2.1.10 pseudomolecules, which were based on version 3 of the genome assembly (Potato Genome Sequencing Consortium Public Data Release).21 Genes reflecting alternative splice sites were designed to represent the exons. In total, the 56 K microarray was designed to include 56,647 predicted genes with 125,924 probes. We scanned the microarray for Cy3 signals with the Genepix 4000B Scanner (Molecular Instruments, LLC, Sunnyvale, CA, USA), and digitized the signals using Nimblescan (Roche NimbleGen, Inc., Madison, WI, USA).
7
Differential DNA Methylation in Obesity
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Raw DNA methylation array data was processed using NimbleScan (Roche NimbleGen) to obtain the statistical significance (P-value) of the differential methylation between the subjects (lean or obese) and their controls for each probe on the array. Probes that overlap with a certain gene feature (e.g., TSS1500 regions defined as between -1,500 bp and +500 bp to TSS) were assigned to the nearest gene using BEDTools [24 (link)]. The P-values for all probes associated with a particular gene in the lean and obese samples were used to derive the statistical significance (P-value) of the differential methylation between lean and obese using the Fisher’s combined probability test [25 ]. Finally, genes with a P-value ≤ 0.05 in the Fisher’s combined probability test were deemed differentially methylated between lean and obese. These genes were further divided into two groups, representing genes that are either hypermethylated in obese or hypomethylated in obese. Functional enrichment of known pathways and Gene Ontology (GO) terms were determined using the Database for Annotation, Visualization and Integrated Discovery (DAVID) web tool [26 (link)].
8
Differential Expression of lncRNAs in Kidney
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Kidney samples from 3 experimental mice and 3 control mice were extracted and used to synthesize double-stranded complementary DNA, which was labeled and hybridized to 8×60 K lncRNA Agilent Genomic Expression Arrays. The gene chips were washed, stained, and then scanned with an Axon GenePix 4000B microarray scanner (Molecular Devices, USA). The raw data were extracted as paired files using the NimbleScan software (version 2.5; Roche NimbleGen, USA). The hierarchical clustering of the differentially expressed lncRNAs was performed using the Cluster 3.0 and Java Treeview (USA). The Gene Ontology (GO) annotations for the microarray genes were downloaded from the NCBI and Gene Ontology databases. A pathway analysis was carried out using the KEGG database.
9
Microarray analysis of B. pseudomallei sigma E mutant
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Images were acquired with Axon GenePix 4000B laser scanner (Molecular Devices) at 5 μm resolution and intensity data were extracted using the software NimbleScan (Roche NimbleGen). Data obtained from hybridizations of two independent RNA preparations of each sample were used for final analysis. Raw microarray data were first LOWESS (Locally Weighted Scatter Plot Smoother) normalized using GeneSpring GX (Agilent) to correct for dye-bias within array followed by median normalization to normalize across all arrays. Finally, the median ratio of probes corresponding to Sanger’s 5935 genes comparing between B. pseudomallei wild type and σE mutant was computed.
10
aCGH Array Data Analysis
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aCGH array was performed as described before.16 (link) NimbleScan and SignalMap softwares were used to analyze the aCGH data (Roche Diagnostics, Mannheim, Germany). Normalized and log2-transformed ratios were used to define gain or loss scoring thresholds.
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