Glume and endosperm material for three biological replicates was harvested from 0 (only glumes), 4, 8, 10, 14, 18, and 24 (glumes and endosperm) DAP; and total RNA was extracted with a Spectrum™ Plant Total RNA Kit (Sigma Aldrich, Steinheim, Germany). RNA integrity was confirmed using the Bioanalyser system (Agilent Technologies). 100ng RNA was used for cRNA synthesis and Cy3-labelling with a Low Input Quick Amp Labelling Kit (Agilent Technologies). Labelling efficiency, and amount and quality of cRNA, were assured using an ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, USA) and Bioanalyser system. 600ng labelled cRNA was used for fragmentation and array loading (Gene Expression Hybridization Kit, Agilent Technologies). Hybridization was done for 17h at 65°C. After washing (Gene Expression Wash Buffer Kit, Agilent Technologies) and drying, arrays were scanned at 5 µm resolution using an Agilent Technologies Scanner G2505C. Resulting images were evaluated (determination of spot intensities, background correction) with Feature Extraction V11.5 (Agilent Technologies).
Bioanalyser system
Manufactured by Agilent Technologies
Sourced in United States
The Bioanalyzer system is a lab equipment product developed by Agilent Technologies. It is a microfluidics-based platform designed for the analysis of DNA, RNA, and proteins. The system integrates sample preparation, separation, detection, and data analysis capabilities in a single automated instrument.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Low input quick amp labelling kit, by Agilent Technologies (1 mentions)
Scanner g2505c, by Agilent Technologies (1 mentions)
Feature extraction v11, by Agilent Technologies (1 mentions)
Gene expression hybridization kit, by Agilent Technologies (1 mentions)
Gene expression wash buffer kit, by Agilent Technologies (1 mentions)
Spectrum plant total rna kit, by Merck Group (1 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Ribo zero kit, by Illumina (1 mentions)
Dnase 1 treatment, by Qiagen (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Trizol plus rna purification kit, by Thermo Fisher Scientific (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
Nucleospin rna plus kit, by Macherey-Nagel (1 mentions)
Rnaqueous micro kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using bioanalyser system
1
RNA Extraction and Microarray Analysis
2
RNA-Seq Library Preparation Protocol
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RNA samples were treated with DNases (AMBION) to remove DNA contaminants, purified with the RNA MEGAclear kit (ThermoFisher), and depleted of ribosomal RNA with the riboZero kit (Illumina). RNA (total, depleted, purified) was checked on the Bioanalyser system (Agilent) for its quality and integrity. cDNA libraries were prepared with samples displaying a RNA integrity number above 7. RNA concentrations were measured using the nanodrop spectrophotometer (Thermo Scientific) and the Qubit fluorometer (Invitrogen). Libraries were prepared with the TruSeq Stranded RNA LT prep kit cDNA synthesis, set A (Illumina) which consists in: (1) RNA fragmentation, (2) 1st strand cDNA synthesis (Reverse transcriptase and random primers), (3) 2nd strand cDNA synthesis (removal of the RNA template and synthesis of a new strand with dUTP), (4) no end repair step, (5) adenylation of 3’ ends, (6) ligation of adapters and (7) enrichment of DNA fragments. Libraries were checked for concentration and quality on DNA chips with the Bioanalyzer Agilent. More precise and accurate quantification was performed with sensitive fluorescent-based quantitation assays ("Quant-It" assays kit and Qubit fluorometer, Invitrogen).
3
RNA Extraction and Quality Control
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Total RNA from the cell culture was isolated from each sample using the miRNeasy mini kit (Qiagen Westburg BV, Leusden, the Netherlands) according to manufacturer's protocol, followed by a DNAse I treatment (Qiagen Inc). Total RNA concentration and quality were measured by means of a BioAnalyser system (Agilent Technologies, Breda, the Netherlands). All sequenced samples were generated from high quality RNA samples having a RIN number above 8.
4
RNA Extraction from Pooled Insect Heads
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RNA was isolated from 12 pooled heads using the TRIzol Plus RNA Purification Kit in conjunction with the PureLink RNA Mini Kit (Life Technologies, Paisley, UK) according to the manufacturer's instructions. Additional steps for ‘On-Column PureLink DNase Treatment During RNA Purification’ were followed. Concentration and integrity of RNA samples were checked using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE) and a bioanalyser system (Agilent Technologies, Santa Clara, CA), respectively. Total RNA obtained was (mean total RNA available for library preparation = 0.855 µg, s.d. = 0.297 µg) of good quality (260/280 ≥ 1.8, RIN values ≥ 8.0 for all samples).
5
Glioblastoma Multiforme RNA Extraction and Microarray Analysis
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Approximately 50 mg of tissues from the initial 54 GBM samples were used to extract total RNA using the RNeasy Lipid Tissue mini kit (Quiagen, CA), following the instructions of the manufacturer. The quality of RNA obtained was checked using a Bioanalyser System (Agilent Technologies, Paolo Alto, CA) using the RNA Nano Chips. Genome U133 Plus 2.0 Expression arrays data processing was done according to the manufacturer recommendations. Normalization was performed using the RMA method.71 Clustering analysis and class comparison using a univariate t‐test were performed using dChip software (http://biosun1.harvard.edu/complab/dchip/ ).72 A p‐value <.005 was used to define differentially expressed genes. In order to compare the gene expression profile of the gliomas with normal brain, we used the gene expression data of five samples of corpus callosum (GSM175855, GSM175856, GSM175857, GSM175858, GSM176050) and five samples of cortex (GSM176049, GSM176344, GSM176345, GSM176346, GSM176347), available in the Gene Expression Omnibus repository (GSE7307).
6
Microarray Analysis of Mouse Intestinal RNA
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Microarray analysis was performed by the Ramaciotti Centre for Gene Function Analysis. Mouse intestinal RNA was isolated using a Nucleospin RNA plus kit (Macherey Nagel, Düren, Germany). RNA quality was measured with an Agilent Bioanalyser system and only RNA with an integrity >7.0 was used for RNA analysis on an Agilent 60K mouse GE array.
7
Microdissecting Rat Oxyntic Mucosa
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Biopsies from rat oxyntic mucosa (n = 6) were mounted in Tissue‐Tek OCT (Sakura, Alpena an den Rijn, The Netherlands) and from each biopsy a 5 µm guiding section was cut, followed by three 16 µm sections which were mounted on membrane slides [Molecular Machines and Industries (MMI), Zurich, Switzerland]. The guiding section was stained for the proliferation marker Ki67 to identify the isthmus zone and placed in the MMI CellCut Plus instrument (with Olympus IX71 inverted microscope) together with the sequential unstained sections from which the tissue was harvested. The isthmus zone was located using the software's serial section function (CellTools, v. 4.0.9) that allowed the operator to transfer information about the cutting area from one stained guiding section to unstained serial sections. RNA was extracted from the microdissected tissues using RNAqueous Micro Kit (Life Technologies, Grand Island, NY, USA); the first incubation was performed with the tubes upside down, to ensure lysis of the harvested tissue adherent to the MMI cap. RNA integrity was measured as a RIN score using the Bioanalyser system (Agilent Technologies, Santa Clara, CA, USA) (see supplementary material, Supplementary materials and methods).
8
Ncoa2 Knockout CD8+ T Cell Transcriptome
9
T cell RNA-seq of Ncoa2 knockout mice
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WT or Ncoa2 fl/fl /CD4 Cre OT1 mice were challenged with MC38-Ova cancer cells for 3 days. CD8 + T cells from the spleens were sorted as described above for RNA extraction with the RNeasy ® mini kit (QIAGEN). RNA quality and quantity were assessed using a BioAnalyser Systems (Agilent Technologies) at Novogene. Selected samples with RNA Integrity Number (RIN) higher than 8.0 were subjected to sequencing library preparation. For RNA-seq data analysis, sequence reads passing quality check were preprocessed for adaptor and polyA removal using trimmomatic v.0.39 and fastp v.0.23.2, followed by mapping to the mouse reference genome mm10, with an index build containing the transcript/gene annotations using STAR v.2.7.9a. Paired-end fragment counts of each gene annotated in RefGene were calculated by the featureCounts function in the Subread package v.1.6.4. To promote the accuracy of the gene expression estimates, a total of 13561 genes with counts per million (CPM) > 0.1 in at least three samples were selected for downstream analysis. Bioconductor package edgeR v.3.32.1 was applied with a design matrix for testing differential expression between Ncoa2 fl/fl CD4 Cre and WT samples based on quasilikelihood F-tests. Statistical p-values were adjusted by Benjamini and Hochberg method. Genes below the false discovery rate of 0.05 were considered significant.
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