After validation of RNA quality with the Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit), 50 ng of total RNA were reverse transcribed following the Ovation PicoSL WTA System (Nugen). Briefly, the resulting double-strand cDNA was used for amplification based on SPIA technology. After purification according to the Nugen protocol, 5 μg of single strand DNA were used for generation of Sens Target DNA using the Ovation Exon Module kit (Nugen). 2.5 μg of Sens Target DNA were fragmented and labelled with biotin using Encore Biotin Module kit (Nugen). After control of fragmentation using a Bioanalyzer 2100, the cDNA was then hybridized to GeneChip® Mouse Gene 1.0 ST (Affymetrix) at 45°C for 17 h. After overnight hybridization, the ChIPs were washed using the fluidic station FS450 following specific protocols (Affymetrix) and scanned using the GCS3000 7G. The scanned images were then analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files) and metrics for Quality Controls. The analysis of some of these metrics and the study of the distribution of raw data show no out-lier experiments. RMA normalization was performed using R and normalized data were subjected to statistical tests.
Encore biotin module kit
Manufactured by Tecan
Sourced in United States
The Encore Biotin Module kit is a laboratory equipment product manufactured by Tecan. It is designed to provide a solution for biotin labeling of nucleic acids. The kit includes the necessary reagents and consumables required for the biotin labeling process. The core function of this product is to facilitate the addition of biotin molecules to nucleic acid samples, enabling their downstream detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ovation pico wta system v2 kit, by Tecan (5 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (4 mentions)
Ovation pico wta system v2, by Tecan (4 mentions)
Expression console software, by Thermo Fisher Scientific (3 mentions)
Agilent rna6000 nano chip kit, by Agilent Technologies (3 mentions)
Ovation picosl wta system, by Tecan (3 mentions)
Ovation exon module kit, by Tecan (3 mentions)
Genechip mouse gene 1.0 st, by Thermo Fisher Scientific (3 mentions)
Fs450, by Thermo Fisher Scientific (3 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (3 mentions)
Genechip fluidics station 450, by Thermo Fisher Scientific (2 mentions)
Human transcriptome arrays 2.0 microarrays, by Thermo Fisher Scientific (2 mentions)
Mouse gene 2.0 st array, by Thermo Fisher Scientific (2 mentions)
Expression console, by Thermo Fisher Scientific (2 mentions)
Nugen picov2 kit, by Tecan (2 mentions)
Gcos software, by Thermo Fisher Scientific (2 mentions)
Genechip platform, by Thermo Fisher Scientific (2 mentions)
Ovation pico wta system, by Tecan (2 mentions)
Genechip command console, by Thermo Fisher Scientific (1 mentions)
Rneasy lipid tissue kit, by Qiagen (1 mentions)
20 protocols using encore biotin module kit
1
Affymetrix Microarray Gene Expression Analysis
2
Affymetrix Gene Expression Profiling
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Biotinylated double-stranded cDNA targets were prepared from 10 ng of total RNA using the NuGEN Ovation Pico WTA System V2 kit (catalog number 3302) followed by the NuGEN Encore Biotin Module Kit (catalog number 4200) according to the manufacturer’s recommendations. Following fragmentation and labeling, 4.55 μg of cDNA was hybridized for 16 h at 45 °C, 60 rpm on Human GeneChip Human Genome U133 plus 2.0 arrays (Affymetrix). The chips were washed and stained in a GeneChip Fluidics Station 450 (Affymetrix) using the FS450_0004 script and scanned with a GeneChip Scanner 3000 7G (Affymetrix) at a resolution of 1.56 μm. Raw data (.CEL intensity files) were extracted from the scanned images using the Affymetrix GeneChip Command Console (AGCC) version 4.0.
3
Gene Expression Analysis of MDS-Derived cDC2 and Monocytes
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RNA was isolated from MDS BM- and NBM-derived cDC2 and slan+ monocytes (5.000-67.000 cells) and amplified using the Ovation Pico WTA System V2 (NuGen, San Carlos, CA) as previously described.11 RNA was labeled with the Encore Biotin Module Kit (NuGEN) and 5 mg of cDNA from each sample was hybridized to Human Transcriptome Arrays 2.0 microarrays (Affymetrix) and signals were scanned by Affymetrix GeneChip Scanner 3000 7G. See the Online Supplementary Appendix for details on data analysis. The microarray data have been deposited in the GEO public database under the accession number: GSE161058.
4
Transcriptomic Profiling of Hematopoietic Cell Populations
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BM HSC(Flt3−CD150+Lin−Sca-1+Kit+), LMPP(Flt3hiLin−Fca-1+Kit+), CLP (Lin−KitloSca-1loFlt3+IL-7Rα+), Flt3+CMP (Flt3+Lin−Sca-1−Kit+FcγR−CD150−CD34+)33 (link), Flt3−CMP (Flt3+Lin−Sca-1−Kit+FcγR−CD150−CD34+)33 (link), EILP (Lin−TCF-1+Thy-1−IL-7Rαneg/lo) and thymus ETP (Lin−KithiCD25−), DN3 (Lin−Kit−CD25+) were isolated by flow cytometric cell sorting. Microarray were conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual by the UPENN Microarray Core Facility. RNA was extracted with Trizol and amplified using Nugen PicoV2 kit (Nugen), and the quality of the RNA was tested on a bioanalyser. Biotinylated cDNA were prepared using Encore Biotin Module kit (Nugen) from 5.5 ug RNA according to the manufacture's instructions. 2.5ug/ul cDNA were hybrizied at 16 hours at 45 C on Affymetrix Mouse Gene 2.0 ST Array. The microarrays were then washed and stained with streptavidin-phycoerythrin. GeneChips were scanned using the GeneArray Scanner 3000 7G. The data were analysed using Affymetrix Expression Console with the default analysis settings. Gene signal values for the arrays were normalized and log2-transformed. Heatmaps were generated using the pheatmap R package (version 1.0.2)49 .
5
Transcriptome Analysis of HERV-V3 Expression
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The cDNA synthesis and amplification steps were performed using 16 ng of RNA with the Ovation Pico WTA System V2 kit (Nugen) according to the manufacturer’s instructions. Five micrograms of amplified purified DNA were fragmented into 50–200 bp fragments and were 3-labeled using the Encore Biotin Module kit (Nugen) according to the manufacturer’s instructions. The HERV-V3 microarrays were hybridised at 50 °C for 18 h in an oven with constant stirring (60 rpm). Washing and staining were carried out according to the protocol provided by the manufacturer, using the GeneChip fluidics station 450 (Affymetrix). The arrays were finally scanned using the GeneChip scanner 3000 7G (Affymetrix) fluorometric scanner. Images (DAT files) were converted to CEL files using GCOS software (Affymetrix) [30 (link)]. The experimental data generated have been filed with the National Center for Biotechnology Information (NCBI) and are available on the GEO DataSets site under access number GSE108239.
6
Mammary Transcriptome Analysis of BPA Exposure
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Total RNA from the mammary glands of animals treated with BPA or vehicle was isolated using the RNeasy Lipid Tissue kit (Qiagen Inc., Valencia CA), which uses a combination of Qiazol followed by column extraction. Next, 50 ng of the isolated RNA was used to generate amplified cDNA using the WT-Ovation Pico RNA Amplification System (NuGEN, San Carlos, CA). Five µg of amplified cDNA from each sample were labeled with biotin using the Encore Biotin Module kit (NuGEN) and subsequently hybridized to the Rat Genome 230 2.0 Chip Arrays (Affymetrix). Eight microarray data sets, which represented 4 BPA-treated and 4 control animals, were obtained for each time point (24 microarrays in total). Raw data were generated by the GeneChip Scanner 3000 using the appropriate software. Pairwise analysis between the two treatment groups was performed using the GeneSifter.net on-line service (vizXlabs, Seattle, WA) and gene expression differences with p<0.05 were identified.
7
HERV-V3 Microarray Protocol: Transcriptome Profiling
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The cDNA synthesis and amplification steps were performed from 16 ng of RNA, using the Ovation Pico WTA System V2 kit (Nugen), in accordance with the manufacturer’s instructions. Five micrograms of purified, amplified cDNA was fragmented into 50–200 bp fragments and were 3′-labeled using the Encore Biotin Module kit (Nugen), in accordance with the manufacturer’s instructions. The HERV-V3 microarrays were hybridized at 50 °C for 18 h in an oven, with constant stirring (60 rpm). Washing and staining were performed using the protocol supplied by the manufacturer, using the GeneChip fluidic station 450 (Affymetrix). The arrays were scanned using the GeneChip fluorometric scanner 3000 7G (Affymetrix). Images (DAT files) were converted to CEL files using the GCOS software (Affymetrix) [22 (link)]. The experimental data generated were deposited in the National Center for Biotechnology Information (NCBI) and are available in the GEO DataSets site, under accession number GSE121352.
8
Microarray Analysis of CP-CML Patient Samples
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Microarray experiments were performed on the BM CD34+/lin- cells of 80 CP-CML patients at diagnosis as well as those who had undergone 12 months of nilotinib treatment. We prepared cDNA starting from the previously extracted RNA (50 ng) using Ovation Pico WTA System V2 kit (NuGEN) and Encore Biotin Module Kit (NuGEN) following the manufacturer’s instructions.
cDNA was hybridized to Affymetrix HTA 2.0 using the Gene Chip platform (Affymetrix, Santa Clara, Ca, USA) and signals were scanned by Affymetrix Gene Chip Scanner 3000 according to the manufacturer’s instructions as described inhttp://dx.doi.org/10.17504/protocols.io.yncfvaw , and in our previous manuscript [18 (link)].
cDNA was hybridized to Affymetrix HTA 2.0 using the Gene Chip platform (Affymetrix, Santa Clara, Ca, USA) and signals were scanned by Affymetrix Gene Chip Scanner 3000 according to the manufacturer’s instructions as described in
9
Microarray Gene Expression Profiling
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After validation of RNA quality with the Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit), 50 ng of total RNA were reverse transcribed following the Ovation PicoSL WTA System (Nugen). Briefly, the resulting double-strand cDNA was used for amplification based on SPIA technology. After purification according to Nugen protocol, 5 µg of single strand DNA was used for generation of Sens Target DNA using Ovation Exon Module kit (Nugen). 2.5 µg of Sens Target DNA were fragmented and labelled with biotin using Encore Biotin Module kit (Nugen). After control of fragmentation using Bioanalyzer 2100, the cDNA was then hybridized to GeneChip Mouse Gene 1.0 ST (Affymetrix) at 45°C for 17 hours. After overnight hybridization, the ChIPs were washed using the fluidic station FS450 following specific protocols (Affymetrix) and scanned using the GCS3000 7G. The scanned images were then analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files) and metrics for Quality Controls. The analysis of some of these metrics and the study of the distribution of raw data show no outlier experiment. RMA normalization was performed using R and normalized data was subjected to statistical tests.
10
Microarray Analysis of Gene Expression
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Microarray was performed in the Microarray Facility Tübingen (Germany). First, total RNA from three animals per time point was amplified using the Ovation Pico WTA System (NuGEN). The Encore Biotin Module Kit (NuGEN) was used for the fragmentation and labeling of cDNA for further analysis. Finally, samples were hybridized using Affymetrix GeneChip Mouse Gene 1.0 ST arrays (Affymetrix). Gene expression data were analyzed using Affymetrix Expression Console software (Affymetrix) and Partek Genomics Suite 6.5 software (Partek). Data were normalized and filtered for transcripts, which were differentially expressed between lesioned and control animals. Significance with p ≤ 0.05 was calculated using analysis of variance (ANOVA).
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