One-ml of TRIzol reagent (Life Technologies, Darmstadt, Germany) was added to each of the punched tissue samples. The resulting suspensions were homogenized by 20 passages through a 22 gauge needle. Chloroform was added and the RNA isolated by phase separation after centrifugation. The RNA-containing upper phases were carefully removed and purified with an RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany) and Genomic DNA eliminated with a DNase step (Qiagen). RNA yield and purity were assessed with a NanoDrop 1000 spectrophotometer (Peqlab, Erlangen, Germany) and samples having optical density 260/280 measurements in the range of 1.8 to 2.2 were kept for further analysis. RNA integrity was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA); only samples with RNA integrity values above 8 were used.
Nanodrop 1000 spectrophotometer
Manufactured by Avantor
Sourced in Germany
The NanoDrop 1000 spectrophotometer is a compact, microvolume UV-Vis spectrophotometer designed for the measurement of nucleic acid and protein concentrations. It utilizes a unique sample retention system that requires only 1-2 microliters of sample to make accurate absorbance measurements. The instrument provides a wide wavelength range and high sensitivity for a variety of life science applications.
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Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (8 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Rna 6000 series 2 nano labchip analysis kit, by Agilent Technologies (2 mentions)
Rneasy midi kit, by Qiagen (2 mentions)
Rneasy rna isolation kit, by Qiagen (2 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Dna prep, by Illumina (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Genuptm total rna kit, by Biotechrabbit (1 mentions)
Lowry assay, by Bio-Rad (1 mentions)
Ion total rna seq kit v2, by Thermo Fisher Scientific (1 mentions)
Ion s5 xl system, by Thermo Fisher Scientific (1 mentions)
Kapa library quantification kit for ion torrent, by Roche (1 mentions)
Dynabeads mrna direct micro kit, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
36 protocols using nanodrop 1000 spectrophotometer
1
RNA Extraction from Tissue Samples
2
Spectral Analysis of Pigment B15:3
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Spectral absorbance of pigment B15:3 (2 μl of an 0.2 mg/ml aqueous pigment dispersion) was measured using the NanoDrop® 1000 spectrophotometer (Peqlab, Erlangen, Germany) according to manufacturer’s instructions.
3
High-quality Genomic DNA Extraction
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To obtain concentrated high-molecular-weight genomic DNA, about 1 cm2 of mycelium was taken and ground in liquid nitrogen with a pre-cooled pistil. Genomic DNA was isolated using the MagAttract HMW DNA Kit following the manufacturer’s protocol and subsequent purification steps with magnetic beads (HMW genomic DNA from fresh or frozen tissue protocol), resulting in a total yield of 1 µg. The final concentration was 9.53 ng/µL, measured with a NanoDrop 1000 spectrophotometer (Peqlab Biotechnologie, GmbH, Erlangen, Germany) and Qubit 4 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) using 1X dsDNA HS Assay Kits. DNA extraction, PCR amplification and Sanger sequencing (using nrITS primers) were performed to evaluate possible contamination and confirm the cultures’ identities. First, a paired-end library was constructed using Illumina DNA Prep (earlier known as Nextera DNA Flex Library Prep) and sequenced on NovaSeq 6000 (NovaSeq SP 150 bp Paired-end Flow Cell, Illumina) at the Biomedical Sequencing Facility (BSF, Vienna, Austria). A total concentration of 0.2 µg was used for Illumina library preparation. To supplement the Illumina data, we prepared several Oxford Nanopore libraries using the SQK-LSK109 kit and sequenced them on a MinION sequencer using R9.4.1 flow cells (altogether, four runs were conducted). The total yield of DNA was in the range of 0.12–0.31 µg.
4
Tissue Collection and RNA Extraction
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Six hours after tDCS, four rats of each stimulation group were deeply anesthetized and decapitated. The brains were rapidly removed and the sensorimotor cortices of each hemisphere were isolated. 20mg cortical tissue of each hemisphere was crushed and stabilized overnight in PurifyLater Tissue Stabilizer (BioEcho, Dormagen, Germany). On the next day, the total RNA was isolated using the GenUPTM Total RNA Kit (Biotechrabbit, Henningsdorf, Germany) according to the manufacturer’s guidelines. RNA quantification was carried out using a NanoDrop-1000 spectrophotometer (Peqlab, Erlangen, Germany), and RNA quality was monitored by agarose gel separation and with the Agilent 2100 Bioanalyzer (Agilent, Böblingen, Germany). All extracted RNA samples were found to be of good quality. RNA integrity numbers (RINs) ranged from 9.8 to 10.
5
Characterization of Extracellular Vesicles
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Human BM-MSC derived EV were lysed in Lämmli buffer, the protein concentration determined with Lowry Assay (Bio-Rad Laboratories GmbH, München, Germany) in a NanoDrop 1000 spectrophotometer (PEQLAB Biotechnologie, Erlangen, Germany) and 10 μg of protein lysate analyzed by 10% SDS-PAGE electrophoresis and Western transfer onto PVDF membrane using standard procedures. Primary antibodies against CD9, CD63, CD81 and Hsp70 were used at 1:1000 and horse reddish peroxidase-coupled (HRP) secondary goat anti-rabbit at 1:20,000 concentrations (EXOAB, System Biosciences, CA, USA). Detection was done with SuperSignal West Pico Chemoluminescence Reagent (Thermo Fisher Scientific, Waltham, MA, USA).
6
RNA-Sequencing of Chicken B Cells
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RNA was isolated from three independent experiments of CVI988 or RB1B infected chicken B cell cultures and sequenced as described [24 (link)]. Briefly, total RNA was extracted using TRIzol reagent (Life Technologies; Carlsbad, CA, USA) in combination with the RNeasy Mini Kit (Qiagen; Hilden, Germany) following the manufacturer’s instructions. Additionally, RNA was treated with DNase using the RNase-Free DNase Set (Qiagen). Subsequently, ERCC ExFold RNA Spike-In mix 1 (Invitrogen; Carlsbad, CA, USA) was added to the total RNA as an internal control and the polyadenylated (poly(A)) RNA fraction was extracted using the Dynabeads mRNA DIRECT Micro kit (Invitrogen). Whole transcriptome libraries were prepared using the Ion Total RNA-Seq Kit v2 (Life Technologies) following the manufacturer’s instructions. Quality and quantity of the nucleic acids was controlled at each step using the NanoDrop 1000 spectrophotometer (Peqlab) or Agilent 2100 Bioanalyzer (Agilent Technologies; Böblingen, Germany) in combination with appropriate chips, respectively. The resulting libraries were finally quantified using the KAPA Library Quantification Kit for Ion Torrent (Kapa Biosystems; Wilmington, MA, USA) on a CFX96 Real-Time PCR Detection System (BioRad Laboratories) and sequenced on an Ion S5XL system (Life Technologies) using the Ion 540 OT2 and Chip kit (Life Technologies).
7
Transcriptome Analysis using Gene 2.1 ST Array
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For transcriptomics analyses, total RNA was isolated using the RNeasy system (QIAGEN, Hilden, Germany). Total RNA concentrations were determined by the NanoDrop-1000 spectrophotometer (PEQLAB, Erlangen, Germany), and RNA quality was assessed by the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) (Figure S5 ). Transcriptome profiling was carried out by the Genomics Core Facility at the Medical University of Vienna (Vienna, Austria) using the Human Gene 2.1 ST Array (Thermo Fisher Scientific). Transcriptome Analysis Console software (v.4.0; Thermo Fisher Scientific) was used for data analysis, for PCA, to determine DEGs, for hierarchical clustering, for scatterplots, and to identify pathways associated with DEGs. Gene lists of DEGs (more than 2-fold change of log2-transormed expression values, p values below 0.05) were analyzed by Cytoscape (v.3.8.5)53 (link) using the ClueGO (v.2.5.7) plug-in.54 (link) Biological process, immune system process, molecular function, and Kyoto Encyclopedia of Genes and Genomes (KEGG) were selected to identify pathways and ontologies.
8
Treg-specific DNA Methylation Analysis
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Genomic DNA was isolated from sorted cells using the NucleoSpin XS kit (Macherey-Nagel) following the manufacturer’s protocol. The DNA concentration was quantified with a Nanodrop 1000 spectrophotometer (Peqlab). Methylation analysis of the TSDR and other Treg-specific epigenetic signature genes was performed using bisulfite sequencing as described previously (28 (link)). Only cells from male mice were used for the methylation analysis.
9
RNA Extraction from Muscle Tissue
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For RNA extraction, the muscle tissue was homogenized mechanically via a steel micropestle (Cat. #6-1062, neoLab, Heidelberg, Germany) in liquid nitrogen. Total RNA was extracted via the Maxwell 16 LEV simplyRNA Tissue Kit (Cat. #AS1280, Promega, Madison, WI) on a Maxwell 16 Instrument (Cat. #AS2000, Promega, Madison, WI) according to the manufacturer’s instructions. RNA concentration was measured with a NanoDrop 1000 Spectrophotometer (Peqlab, Erlangen, Germany).
10
Epigenetic Profiling of Thymocyte Subsets
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Genomic DNA was isolated from sorted CD4SP thymocyte subsets using the DNeasy® Blood & Tissue Kit (Qiagen) and concentrated using the DNA Clean & Concentrator Kit (Zymo Research), both following the manufacturers' protocols. The DNA concentration was quantified with a Nanodrop 1000 spectrophotometer (Peqlab). Methylation analysis of the TSDR and other Treg cell-specific epigenetic signature genes was performed using bisulfite sequencing as described before (27 (link)). Exclusively, cells from male mice were used for the methylation analysis. For each CD4SP thymocyte subset, cells were pooled from 4 to 6 independent experiments to reach sufficient cell numbers for the methylation analyses.
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