Microarray analysis was performed on liver samples from the mice fed the three different diets as well as on Hepa1‐6 hepatoma and differentiated 3T3‐L1 adipocyte cells incubated with different fatty acids. RNA was purified with RNeasy Minikit columns (Qiagen) and analyzed for quality with RNA 6000 Nano chips on the Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands). One microgram of RNA was used for cDNA synthesis using the First Strand cDNA synthesis kit (Thermo Scientific). Purified RNA (100 ng) was labeled with the Ambion WT expression kit (Invitrogen) and hybridized to an Affymetrix Mouse Gene 1.1 ST array plate (mouse liver, Hepa1‐6 cells) or 2.1 ST array plate (3T3‐L1 adipocytes) (Affymetrix, Santa Clara, CA). Hybridization, washing, and scanning were carried out on an Affymetrix GeneTitan platform. Scans of the Affymetrix arrays were processed using packages from the Bioconductor project. Arrays were normalized using the robust multi‐array average method.46 , 47 Probe sets were defined by assigning probes to unique gene identifiers, for example, Entrez ID.48 For the Hepa1‐6 and 3T3‐L1 cells, the total gene set (24 973 probe sets) was filtered to only include genes with mean signal >20, yielding 10 379 and 11 504 genes, respectively. Microarray data were submitted to the Gene Expression Omnibus (accession number pending).
Rna 6000 nano chip
Manufactured by Agilent Technologies
Sourced in United States, Netherlands, Germany, Canada, United Kingdom
The RNA 6000 Nano Chip is a lab equipment product designed for the analysis of RNA samples. It provides a platform for the detection and quantification of RNA molecules within a sample. The chip utilizes microfluidic technology to perform electrophoretic separations, allowing for the assessment of RNA integrity and concentration.
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Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (162 mentions)
Trizol reagent, by Thermo Fisher Scientific (69 mentions)
2100 bioanalyzer, by Agilent Technologies (66 mentions)
Nextseq 500, by Illumina (40 mentions)
Rneasy mini kit, by Qiagen (33 mentions)
Nd 2000 spectrophotometer, by Thermo Fisher Scientific (29 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (28 mentions)
Quantseq 3 mrna seq library prep kit, by Lexogen (22 mentions)
Trizol, by Thermo Fisher Scientific (21 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (19 mentions)
Bioanalyzer, by Agilent Technologies (14 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (14 mentions)
Agilent 2100, by Agilent Technologies (14 mentions)
Allprep dna rna mini kit, by Qiagen (12 mentions)
Hiseq 2500, by Illumina (12 mentions)
Rneasy kit, by Qiagen (10 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (10 mentions)
Nanodrop, by Thermo Fisher Scientific (10 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (9 mentions)
Nanodrop spectrophotometer, by Agilent Technologies (8 mentions)
369 protocols using rna 6000 nano chip
1
Microarray analysis of fatty acid effects
2
Microarray Transcriptional Profiling of Mouse Colon
3
Transcriptomic Profiling of Fatty Acid Effects
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Microarray analysis was performed on Hepa1-6 hepatoma cells incubated with different fatty acids. RNA was purified with RNeasy Minikit columns (Qiagen) and analyzed for quality with RNA 6000 Nano chips on the Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands). One microgram of RNA was used for cDNA synthesis using the First Strand cDNA synthesis kit (Thermo Scientific). Purified RNA (100 ng) was labeled with the Ambion WT expression kit (Invitrogen) and hybridized to an Affymetrix Mouse Gene 1.1 ST array plate (Affymetrix, Santa Clara, CA). Hybridization, washing, and scanning were carried out on an Affymetrix GeneTitan platform. Scans of the Affymetrix arrays were processed using packages from the Bioconductor project. Arrays were normalized using the robust multi-array average method [27 (link),28 (link)]. Probe sets were defined by assigning probes to unique gene identifiers, e.g., Entrez ID [29 (link)]. The total gene set (24,973 probe sets) was filtered to only include genes with mean signal > 20, yielding 10,379 genes. Microarray data were submitted to the Gene Expression Omnibus (accession number pending).
4
RNA-seq Analysis of Skeletal Muscle
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Frozen skeletal muscle (8–25 mg) was homogenized on ice in 500 µL of Trizol for RNA extraction using standard protocols (Lee et al., 2020 (link)). RNA quality was recorded by the RNA integrity number (RIN) using the Agilent RNA 6000 Nano chips. Healthy muscle RNA samples with RIN above 7 and DMD muscle RNA samples with RIN above 4 were used to prepare cDNA libraries with ribosomal RNA depletion using the KAPA RiboErase Kit (HMR) (Roche). About 50 million 150-151 bp paired-end RNA sequencing (RNAseq) reads were generated per RNA sample using Illumina Novaseq 6000 S4. Sequencing reads were aligned to GRCh38 (Ensembl 105, Gencode v39) using STAR 2.6.0c (Dobin et al., 2012 (link); Lee et al., 2020 (link)). Data quality control included alignment metrics (ribosomal and globin RNA, aligned and unmapped reads, sequencing depth), hierarchical clustering, principal component analysis and Pearson correlation.
5
Total RNA Extraction from Leaf Tissue
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Total RNA was extracted from 100 mg leaf tissue with RNeasy Plant Mini Kit (Cat # 74904, Qiagen, Hilden, Germany) as per manufacturer’s guidelines. Leaf tissue from the third tier of rosette was harvested, and leaves from three different plants were pooled for each biological replicate. For each treatment, two biological replicates were sampled. The pooled sample was homogenized in liquid nitrogen into a fine powder, and the total RNA was extracted. Following total RNA isolation, DNase digestion (Cat # 79254 RNeasy/QlAamp columns, Qiagen, Hilden, Germany) was performed to remove contaminating DNA, according to the manufacturer’s instructions. RNA integrity was analyzed using the Agilent 2100 Bioanalyzer (Agilent Technologies, California, USA) with RNA 6000 Nano Chips (Cat # 5067-1511, Agilent Technologies, California, USA), following the manufacturer’s protocol. RNA integrity numbers (RIN) ranging from 7.0 to 7.5 were used for the microarray experiment.
6
Liver and Caecum RNA Isolation and Analysis
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A 1/4 portion of the left liver lobe and the empty caecum were stored in RNAlaterTM stabilization solution (Invitrogen, Carlsbad, CA, USA) at −80 °C until further analysis. Total RNA was extracted from the liver with the RNAeasy Plus Mini Kit (Qiagen, Hilden, Germany). RNA integrity and concentration were checked with RNA 6000 Nano chips on an Agilent 2100 bioanalyser (Agilent Technologies, Amsterdam, The Netherlands). Total RNA (10 µg per reaction) was reverse-transcribed into complementary DNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Thermofisher Scientific, Foster City, CA, USA) according to the manufacturer’s instructions. Real-time qPCR was performed on an Applied biosystem step one plus machine. The relative gene expressions were normalized to two housekeeping genes: gapdh and actb (glyceraldehyde-3-phosphate dehydrogenase, actin beta), chosen based on results obtained from TaqMan mouse endogenous control arrays (Applied biosystems). The HR_CD samples were used as reference for the relative expression of genes.
7
Comprehensive RNA Isolation from Mouse Tissues
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Total RNA was isolated from FCx, Hp, and lumbar SC tissues from 6-month-old mice [Wt mice, n = 4 (two males and two females); Tg mice, n = 4 (two males and two females)] using TRIzol reagent (15596–018; Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. RNA samples were digested with the RNase-free DNase Set (79254; Qiagen, Hilden, Germany) to remove genomic DNA and further purified using the RNeasy Kit (74104; Qiagen). RNA sample quality was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) using RNA 6000 NanoChips (5067–1511; Agilent Technologies). RNA samples with an RNA integrity number (RIN) higher than 7 were used for further analysis, including qRT-PCR and RNA sequencing.
8
RNA-seq Library Preparation and Sequencing
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RNA was extracted using the RNeasy Mini Kit (QIAGEN), including the optional DNase step to prevent genomic DNA contamination. RNA integrity was assessed using RNA 6000 Nano Chips on a 2100 Bioanalyser (Agilent). Only samples with non-degraded RNA (RIN ≥ 7) were used for sequencing. Sequencing of RNA samples was performed by deCODE Genetics, Iceland. Preparation of indexed cDNA sequencing libraries was carried out using the TruSeq poly-A mRNA method (Illumina). Briefly, poly-A mRNA transcripts were captured from total RNA using poly-T beads, before cDNA was generated using random hexamer priming. Paired-end sequencing (2 × 100 cycles) of indexed cDNA libraries was then carried out on a HiSeq 2500 machine (Illumina), generating at least 50 million reads (101 base pairs) per sample. Sequencing was performed using v4 SBS and Cluster Kits (Illumina). One sample failed the library generation step and was excluded from the study. After sequencing the indexed samples were demultiplexed before generation of FASTQ files for analysis. Raw data files are available from the European Nucleotide Archive (http://www.ebi.ac.uk/ena ) under the study accession number PRJEB12995.
9
Dorsal Root Ganglia Transcriptomics
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Two hours after the end of the behavioural study (D19), rats were deeply anaesthetized with pentobarbital (60 mg/kg i.p; Ceva Santé Animale, Libourne, France) and decapitated (n = 4 vincristine-morphine, n = 4 vincristine-oxycodone, n = 4 vincristine-saline treated rats; and n = 4 saline-morphine, n = 4 saline-oxycodone, n = 4 saline-saline rats). Lumbar dorsal root ganglia (DRGs) L2 through L5 were dissected and immediately frozen on dry ice. Tissues were kept at −80°C until homogenisation. Total RNA from individually dissected frozen DRGs was extracted and treated with DNAse using the RNeasy® Lipid Tissue Mini kit (Qiagen, Courtaboeuf, France), according to the manufacturer's protocol. The quality and quantity of each RNA sample was checked using an Agilent 2100 Bioanalyzer with RNA 6000 NanoChips (Agilent Technologies, Massy, France). Five hundred ng of RNA were sent to the Integragen Illumina microarray facility (Integragen, Evry, France, www.integragen.com ) for sample processing. Illumina pangenomic rat microarrays (RatRef-12 Expression BeadChips), including 22,517 probes, were also performed at the Integragen Illumina microarray facility (Evry, France), according to the Illumina procedures.
10
Peripheral Blood PBMC Isolation and RNA Extraction
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A total of 20 mL of peripheral blood was collected and used for the isolation of PBMCs by discontinuous gradient density centrifugation on a Ficoll-Hypaque cushion (Sigma, St. Louis, MO). Total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. RNA concentrations and ratios were checked using a NanoDrop ND-1000 spectrophotometer (NanoDrop Products, Wilmington, DE), and the RNA integrity was assessed by microfluidic electrophoresis using a 2100 Bioanalyzer and RNA 6000 nanochips (Agilent Technologies, Santa Clara, CA). We used only samples that exhibited median RNA integrity number (RIN) ≥ 9.0.
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