Total RNA was isolated from the VOR in vitro q24h multidose simulation study cell pellets (described above) using the RNeasy minikit (Qiagen, Valencia, CA) according to the manufacturer’s protocol with modifications described above. RNA samples were quantitated using the Qubit RNA high-sensitivity (HS) assay kit and the Qubit 3.0 fluorometer (Life Technologies, Carlsbad, CA). RNA (30 ng) was converted to cDNA using the SuperScript Vilo kit (Life Technologies, Carlsbad, CA) per the manufacturer’s protocol. Library barcoding, preparation, and sequencing were carried out per the manufacturer’s protocols using the following Ion Torrent kits on the Ion Chef Prep station and the Ion Torrent S5-XL (Life Technologies, Carlsbad, CA): Ion AmpliSeq transcriptome human gene expression panel Chef-ready kit (catalog no. A31446), Ion 540 chip kit (catalog no. A27766) and Ion 540 Kit-Chef (catalog no. A30011) (Life Technologies, Carlsbad, CA). Eight samples per 540 chip were sequenced with 8 to 10 million reads/sample. Sequencing from the AmpliSeq analysis was analyzed using Partek Flow software to evaluate genes modulated with RPM of >70, an FDR of <0.1, and a VOR/DMSO ratio of >1.5 or <0.75 for each time point.
Qubit rna high sensitivity assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Qubit RNA High Sensitivity Assay Kit is a fluorometric-based solution for quantifying low concentrations of RNA samples. The kit provides reagents and protocols for accurate measurement of RNA concentrations ranging from 5 pg/μL to 100 ng/μL.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (11 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (9 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (6 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
2100 bioanalyzer, by Agilent Technologies (4 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (3 mentions)
2200 tapestation system, by Agilent Technologies (3 mentions)
Truseq stranded mrna library prep kit, by Illumina (3 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Nextseq 550 sequencer, by Illumina (3 mentions)
Direct zol rna microprep kit, by Zymo Research (2 mentions)
Trizol, by Zymo Research (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Rna 6000 pico chip, by Agilent Technologies (2 mentions)
Rna 6000 nano kit, by Agilent Technologies (2 mentions)
Rnase free dnase 1, by Qiagen (2 mentions)
60 protocols using qubit rna high sensitivity assay kit
1
Transcriptome Profiling of VOR Simulation
2
FMDV RNA Extraction and Quantification
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Total RNA was extracted from 460 μl of cell culture virus isolate or original suspension [consisting of 10% tissue suspensions generated in M25 phosphate buffer (35 mM Na2HPO4.2H2O; 5.7 mM KH2PO4; pH 7.6; made in-house)] using RNeasy MiniKit (Qiagen) according to manufacturer’s instructions. Total RNA was eluted in 50 μl of nuclease-free water and quantified using the Qubit RNA High Sensitivity (HS) Assay Kit (Life Technologies). FMDV-specific RNA was detected using an FMDV-specific real-time RT-qPCR as described previously (Table
2 )
[24 (link)] and quantified using an RNA standard derived from O/UKG/35/2001.
[24 (link)] and quantified using an RNA standard derived from O/UKG/35/2001.
3
Transcriptome Analysis of Leaf Samples
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Leaf samples were ground in liquid nitrogen. Total RNAs were extracted using the RNeasy mini kit (QIAGEN) following the manufacturer’s protocols. RNA integrity and concentration were determined using a 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) and a Qubit® RNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Only RNAs with RIN (RNA Integrity Number) greater than 8 were used for the next steps. For each sample, 100 ng of total RNA from each sample was used in the Ribo-Zero rRNA removal kit (Illumina, San Diego, CA). Subsequently, the rRNA-depleted RNA was processed using the TruSeq Stranded RNA library preparation kit (Illumina, San Diego, CA). The libraries were evaluated using Qubit® DNA Broad-range Assay Kit and 2100 Bioanalyzer. Individual libraries were uniquely barcoded, multiplexed, and paired-end sequenced (2 × 150 bp) on the Illumina NextSeq 500 sequencer (NextSeq 500 Control Software v.4.0.2) at the Instituto Tecnológico Vale, Belém, Pará, Brazil with the High Output kit v.2.5 (300 cycles). All raw reads generated from this study were deposited in the Short Read Archive (SRA) of NCBI under accession number PRJNA645405.
4
RNA-Seq Analysis of H9c2 Cell Differentiation
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35 × 103 to 1 × 106 of H9c2 cells were plated in 25 cm2 tissue culture flasks at 0, 7, and 14 days and induced to differentiation conditions as described above. Cells were treated directly with TRIzol™ Reagent (Invitrogen), and RNA was quantified with a Qubit RNA High Sensitivity assay kit (Thermo Fisher Scientific). Sequencing library preparation was carried out using cDNA synthesis for next-generation sequencing (NGS) performed by TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA, USA). Paired-end sequencing (2 × 75 bp reads) was done by three biological replicates for each of the three points. Library sequencing was performed using a MiSeq Reagent Kit v3 in a MiSeq Sequencer (Illumina) following the manufacturer’s instructions.
5
Quantifying RNAi Effector Expression
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RNAi effector expression was compared between worms maintained in RPMI and CS50 by extracting mRNA from 20 worms/replicate after one week as described above. RNA concentrations were normalised using the Qubit® RNA High Sensitivity Assay Kit (ThermoFisher Scientific) prior to reverse transcription (as above). qPCRs were run using 2 μl cDNA (diluted 1:1) in a 10 μl reaction with 5 μl SensiFAST SBYR No-ROX Kit (Bioline) and 0.1 μM of each primer. ΔCt values were obtained using half of the Pfaffl equation (Pfaffl, 2001 (link)) with values transformed, via log2, to normalise expression.
6
FFPE Tumor RNA Extraction and Analysis
7
Extracting and Sequencing mRNA from Samples
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RNA was extracted from samples using the ZymoBIOMICS RNA Miniprep Kit (Zymo Research, Irvine, CA, United States) according to the manufacturer’s protocol with the following exceptions: 1 volume of lysis buffer was used, the optional DNase I treatment was completed, and extracts were eluted with 50 uL of DNase/RNase free water. After extraction, quantification was conducted using an Invitrogen Qubit 4 Fluorometer and Qubit RNA High Sensitivity Assay Kit (ThermoFisher Scientific, Waltham, MA, United States).
RNA was then processed to make MT libraries using the NEBNext Ultra II RNA Library Prep Kit for use with Illumina (New England Biolabs, Ipswich, MA, United States), which utilizes a random priming approach for reverse transcription. The protocol specifically for use with purified mRNA was followed. Depletion of rRNA was not performed.
Libraries were quality checked using an Agilent 2100 Bioanalyzer and DNA High Sensitivity kit. Results from these steps were used to pool samples in an equimolar ratio. The pool was then gel purified using a 2% agarose gel and the Qiagen QIAquick gel extraction kit (Qiagen, Germantown, MD, United States). Following purification, the pool was sequenced using an Illumina NextSeq 550 platform to produce 2 × 150 bp reads by Wright Labs LLC (Huntingdon, PA, United States).
RNA was then processed to make MT libraries using the NEBNext Ultra II RNA Library Prep Kit for use with Illumina (New England Biolabs, Ipswich, MA, United States), which utilizes a random priming approach for reverse transcription. The protocol specifically for use with purified mRNA was followed. Depletion of rRNA was not performed.
Libraries were quality checked using an Agilent 2100 Bioanalyzer and DNA High Sensitivity kit. Results from these steps were used to pool samples in an equimolar ratio. The pool was then gel purified using a 2% agarose gel and the Qiagen QIAquick gel extraction kit (Qiagen, Germantown, MD, United States). Following purification, the pool was sequenced using an Illumina NextSeq 550 platform to produce 2 × 150 bp reads by Wright Labs LLC (Huntingdon, PA, United States).
8
Leaf RNA Extraction with Eurogold Kit
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Total RNA was extracted from 100 mg of the fresh leaf using the Eurogold TriFastTM kit (EuroClone, Milan, Italy), following the manufacturer’s instructions. RNA quantification was performed with Qubit RNA High Sensitivity Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).
9
RNA-seq analysis of breast cancer cells
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MDA-MB-231 and HCC1806 cells were seeded into 6-well plates and treated the following day as indicated for 72 h. Total cell RNA from 3 independent experiments was extracted using PureLink RNA Mini Kit (ThermoFisher Scientific) with on-column DNAse treatment according to manufacturer’s instructions. The quality and integrity of the total RNA extracted was determined using TapeStation 2200 system (Agilent Technologies, VIC) and Qubit RNA High Sensitivity assay kit (ThermoFisher Scientific). 500 ng total RNA was used for library preparation according to manufacturer’s instructions (QuantSeq 3' mRNA-Seq FWD, Lexogen). Indexed libraries were pooled and sequenced on a NextSeq500 (Illumina). Briefly, the library was generated with an oligo-dT containing the Illumina Read2 linker and a random forward primer containing the Illumina Read1 linker. The library was then amplified with PCR primers containing sample indices and the Illumina clustering sequences. Five to fifteen million single-end 75 bp reads were generated per sample.
10
RNA Isolation, Sequencing Library Preparation
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Total RNA was isolated using the PicoPure RNA Isolation Kit (Life Technologies) and DNA removal was performed using the RNase-free DNase Set (Qiagen). Extracted RNA quantity was measured by Qubit RNA High Sensitivity Assay Kit (Thermo Fisher Scientific) and quality was assessed by running the samples on RNA ScreenTape Assay on the 2200 TapeStation System (Agilent Technologies) or RNA 6000 Nano Kit on the 2100 Bioanalyzer (Agilent Technologies). Only RNA with sufficient RNA integrity numbers (RIN; at least seven and mostly greater than eight) were used to generate libraries. cDNA sequencing libraries were prepared using the TruSeq RNA Access Library Prep Kit (Illumina) according to the protocols provided by the manufacturer. Using the KAPA Illumina Library Quantification Kits, library pools were quantified on the Eco Real-Time PCR Instrument. All libraries were sequenced on the Illumina HiSeq 2500 platform using paired-end cluster generation and 2 × 126 cycles.
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