Total RNA was isolated using Qiagen RNeasy kit (Qiagen), and 500 ng of total RNA was used to synthesize dscDNA. Biotin-labeled RNA was generated using the TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip (Epicentre), according to the manufacturers’ instructions, and hybridized to the HumanHT-12 v4 Expression BeadChip (Illumina), which contains more than 47,000 probes derived from the NCBI RefSeq Release 38 and other sources, at 58°C for 16 h. The chip was washed, stained with streptavadin-Cy3, and scanned with the Illumina BeadStation 500 and BeadScan. Using the Illumina's GenomeStudio software, we normalized the signals using the “cubic spline algorithm” that assumes that the distribution of transcript abundance is similar in all samples, according to the method proposed by Workman et al. [30 ]. The background signal was removed using the “detection p-value algorithm” to remove targets with signal intensities equal or lower than that of irrelevant probes (with no known targets in the human genome but thermodynamically similar to the relevant probes). Common and unique sets of genes and enrichment analysis were performed using the MetaCore Software (Thompson Reuters) as previously reported [14 (link)]. The microarray original data have been submitted to Gene Expression Omnibus (GEO) database (Accession number: GSE79548).
Beadscan
Manufactured by Illumina
Sourced in United States
The BeadScan is a high-throughput imaging system designed for Illumina's BeadArray technology. It is capable of rapid and accurate scanning of Illumina's BeadChips, which are used for a variety of genomic and genetic analysis applications.
Automatically generated - may contain errors
Lab products found in correlation
Humanht 12 v4 expression beadchip, by Illumina (4 mentions)
Beadarray reader, by Illumina (4 mentions)
Targetamp nano labeling kit for expression beadchip, by Illumina (3 mentions)
Beadstation 500, by Illumina (3 mentions)
Rneasy kit, by Qiagen (3 mentions)
Beadstudio, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Ambion total rna amplification kit, by Thermo Fisher Scientific (2 mentions)
Beadstudio software, by Illumina (2 mentions)
Humanht12 v4 beadchips, by Illumina (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Genomestudio software, by Illumina (1 mentions)
Infinium humanmethylation27 beadchip, by Illumina (1 mentions)
Iscan reader, by Illumina (1 mentions)
Genomestudio methylation software, by Illumina (1 mentions)
Infinium humanmethylation450 beadchip, by Illumina (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Humanht 12 v4 expression beadchip kit, by Illumina (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
17 protocols using beadscan
1
Illumina Expression BeadChip Profiling
2
Microarray Analysis of Gene Expression
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Microarray analysis was performed and analyzed at the Stanley S. Scott Cancer Center's Translational Genomics Core at LSUHSC. Total RNA was isolated using Qiagen RNeasy kit (Qiagen), and 500 ng of total RNA was used to synthesize dscDNA. Biotin-labeled RNA was generated using the TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip (Epicentre), and hybridized to the HumanHT-12 v4 Expression BeadChip (Illumina) at 58°C for 16 h. The chip was washed, stained with streptavadin-Cy3, and scanned with the Illumina BeadStation 500 and BeadScan. Using the Illumina's GenomeStudio software, we normalized the signals using the “cubic spline algorithm” that assumes that the distribution of transcript abundance is similar in all samples. The background signal was removed using the “detection p-value algorithm” to remove targets with signal intensities equal or lower than that of irrelevant probes (with no known targets in the human genome but thermodynamically similar to the relevant probes). The microarray experiments were performed twice for each group and the average values were used for analysis. Common and unique sets of genes and enrichment analysis were performed using the MetaCore Software (Thompson Reuters). The microarray original data have been submitted to Gene Expression Omnibus (GEO) database (Accession number: GSE90038).
3
Genome-wide DNA Methylation Profiling
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High-molecular weight DNA was extracted from fresh frozen tissue samples using phenol–chloroform, followed by dialysis. Genome-wide CpG methylation profiling was performed on lung, stomach and kidney samples using the Infinium HumanMethylation27 BeadChip (Illumina, San Diego, CA), and on breast and liver samples using the Infinium HumanMethylation450 BeadChip (Illumina) (29 (link)). Five-hundred-nanogram samples of DNA were subjected to bisulfite conversion using an EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA). After hybridization, the specifically hybridized DNA was fluorescence-labeled by a single-base extension reaction and detected using a BeadScan or iScan reader (Illumina) in accordance with the manufacturer’s protocol. The data were then assembled using GenomeStudio methylation software (Illumina). At each CpG site, the ratio of the fluorescence signal was measured using a methylated probe relative to the sum of the methylated and unmethylated probes, i.e. the so-called β-value, which ranges from 0.00 to 1.00, reflecting the methylation level of an individual CpG site.
4
Illumina Expression Array Preparation
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Expression arrays were prepared and analyzed as described previously in [25 ]. A total of 330 ng of RNA was used to prepare labeled complementary RNA (cRNA) with the Illumina TotalPrep RNA Amplification Kit (Ambion Inc., TX, USA), according to the manufacturer’s protocol with an overnight incubation. The 2100 Bioanalyzer (Agilent Technologies) was used to determine the quality and concentration of the obtained cRNA. The samples were hybridized to HumanHT-12 v4 expression BeadChip (cat. no. BD-103-0204, Illumina Inc., CA, USA) according to the Illumina protocol using 1500 ng of labeled cRNA for each sample. The experiment was performed at the Department of Biotechnology, University of Tartu. The samples were applied randomly on the BeadChips and scanning was performed with BeadScan (Illumina Inc., CA, USA).
5
Illumina RNA Expression Profiling
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Labelled cRNA was prepared from 330 ng of total RNA using the Illumina TotalPrep RNA Amplification Kit (Ambion Inc., TX, USA) with overnight incubation according to the manufacturer’s protocol. The quality of the labelled cRNA was determined using a 2100 Bioanalyzer (Agilent Technologies). In total, 1500 ng of labelled cRNA was hybridised overnight to a HumanHT-12 v4 Expression BeadChip (Cat no. BD-103-0204, Illumina Inc., CA, USA), according to the Illumina protocol, in the Core Facility of the Department of Biotechnology of the University of Tartu. Samples were assigned to the arrays in a randomised order. The chips were scanned using Beadscan (Illumina Inc.).
6
Microarray Analysis for Gene Expression
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Microarray analysis was performed and analyzed at the Stanley S. Scott Cancer Center's Translational Genomics Core at LSUHSC. Total RNA was isolated using Qiagen RNeasy kit (Qiagen), and 500 ng of total RNA was used to synthesize dscDNA. Biotin-labeled RNA was generated using the TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip (Epicentre), and hybridized to the HumanHT-12 v4 Expression BeadChip (Illumina) at 58°C for 16 h. The chip was washed, stained with streptavadin-Cy3, and scanned with the Illumina BeadStation 500 and BeadScan. Using the Illumina's GenomeStudio software, we normalized the signals using the “cubic spline algorithm” that assumes that the distribution of transcript abundance is similar in all samples. The background signal was removed using the “detection p-value algorithm” to remove targets with signal intensities equal or lower than that of irrelevant probes (with no known targets in the human genome but thermodynamically similar to the relevant probes). The microarray experiments were performed twice for each group and the average values were used for analysis. The enrichment analysis were performed using the MetaCore Software (Thompson Reuters). The microarray original data have been submitted to Gene Expression Omnibus (GEO) database (Accession number: GSE90039).
7
Microarray Analysis of Whole Transcriptome
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Total RNA was isolated by using the Nucleospin® RNA kit protocol (Macherey–Nagel, Germany). Five hundred nanograms of total RNA was amplified using the Ambion total RNA amplification kit (Ambion, Carlsbad, CA), followed by hybridization of the synthesized cRNA with the Human HT-12 v4 Expression BeadChip kit (Illumina, San Diego, CA, USA), in accordance with the manufacturer’s protocol. Scanning of the chip was performed using Illumina BeadScan and BeadStudio (version 3.1). Data were processed with a package from Bioconductor (lumi) [23 (link), 24 (link)] using the free software environment R (http://www.r-project.org/ ). The microarray data has been registered in gene expression omnibus (https://www.ncbi.nlm.nih.gov/geo/ ) in NCBI (GSE95308).
8
Molecular Profiling of Tumor Samples
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In the case series obtained at the INT, representative frozen samples containing >60% of tumor cells were submitted to molecular analyses. Tissue was pulverised using a Mikrodismembrator (Braun Biotech International, Melsungen, Germany). Total RNA was extracted with the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer instructions and processed for microarray hybridisation by the INT Functional Genomics Core Facility. Briefly, 600 ng of RNA was amplified using the Illumina Human_v2 MicroRNA expression profiling kit based on the DASL (cDNA-mediated Annealing, Selection, Extension and Ligation) assay according to the manufacturer's recommendations (Illumina Inc., San Diego, CA, USA), fluorescence-labeled, and then hybridised to the Illumina miRNA BeadChip, which allows analysis of 1146 sequences (representing 97% of validated human miRNAs described in miRBase database version 12.0).
The Illumina BeadArray Reader (San Diego, CA, USA) was used for scanning the arrays, and the Illumina BeadScan (San Diego, CA, USA) software was used for image acquisition and recovery of primary signals.
The Illumina BeadArray Reader (San Diego, CA, USA) was used for scanning the arrays, and the Illumina BeadScan (San Diego, CA, USA) software was used for image acquisition and recovery of primary signals.
9
Genome-Wide Expression Analysis of SOCS3
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Total RNA was extracted from 8348SOCS3 cells and 8348plv cells using Trizol reagent. Genome expression analysis was performed using an Illumina Human HT-12 v4 BeadChip (Illumina, San Diego, CA, USA) at the Beijing Qian Zhao Xing Ye Biological Technology Co., Ltd. (Beijing, China). The beadchips were scanned on the Illumina Bead Array 500GX Reader using Illumina BeadScan image data acquisition software. Illumina BeadStudio software was used for preliminary data analysis. The preliminary data were normalized using sample averages; the sample intensities were scaled by a factor equal to the ratio of average intensity of a sample to the average intensity of the given sample [8 (link)]. 8348SOCS3 cells and 8348plv cells were regarded as the given sample. Each sample was repeated three times. An Illumina custom algorithm was used to compare 8348SOCS3 cells with 8348plv cells. A difference score for a probe (diff score) indicates differential gene expression between the two groups. For each gene, the diff scores of corresponding probes were averaged. The results were validated using qRT-PCR.
10
Illumina Gene Expression Profiling
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Five hundred nanograms of total RNA was amplified using the Illumina TotalPrep RNA amplification kit (Ambion), according to manufacturer’s instructions. cRNA was hybridized to Sentrix Human-6 Expression BeadChips v3 (Illumina), according to standard Illumina protocols. Chips scans were processed using the Illumina BeadScan and BeadStudio software packages, and summarized data were generated in BeadStudio (version 3.4). We used the lumi (Du et al. 2008 (link)) and Limma (Smyth 2004 (link)) packages for normalization and differential analysis of detected intensities by individual probes. In the comparison of gene expression levels with other platforms, we averaged all the probe signals to the corresponding gene. As for the THP-1 and HeLa profiles (100%:0% and 0% and 100%), we used the same data obtained by Kanamori-Katayama et al. (2011) (link) (deposited in GEO as GSE28148).
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