Total RNA was extracted from CD4+ T cells isolated from the liver, lung, and spleen using the TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and the Qubit® RNA Assay Kit in a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA). RNase-free DNase I (Invitrogen, Carlsbad, CA, USA) was used to digest potential genomic deoxyribonucleic acid (DNA). The digested products were then purified using magnetic beads (Axygen, Union City, CA, USA) to eliminate residual DNA and DNase. The concentrate of the extracted RNA was analyzed with a Qubit® RNA Assay Kit in Qubit®2.0 Fluorometer (Life Technologies, CA, USA). RNA purity and integrity were assessed using a NanoPhotometer® spectrophotometer (Implen, CA, USA) and an RNA 6000 Nano Assay Kit of the Agilent 2100 Bioanalyzer system (Agilent Technologies, CA, USA). Total RNA of all samples had a concentration ≥ 200 ng/mL, a mass ≥ 10 mg, and an RNA integrity number (RIN) ≥ 8.0.
Qubit rna assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Canada, China
The Qubit® RNA Assay Kit is a quantitative and sensitive fluorescence-based assay designed for measuring the concentration of RNA in solution. The kit provides a simple and accurate method to determine the RNA concentration in a sample.
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Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (367 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (344 mentions)
Nanophotometer spectrophotometer, by Implen (225 mentions)
Trizol reagent, by Thermo Fisher Scientific (213 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (199 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (161 mentions)
Qubit 2.0 flurometer, by Thermo Fisher Scientific (121 mentions)
Nanophotometer, by Implen (89 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (71 mentions)
Trizol, by Thermo Fisher Scientific (50 mentions)
2100 bioanalyzer, by Agilent Technologies (47 mentions)
Hiseq platform, by Illumina (44 mentions)
Hiseq 2500 platform, by Illumina (44 mentions)
Rneasy mini kit, by Qiagen (29 mentions)
Truseq pe cluster kit v3 cbot hs, by Illumina (28 mentions)
Hiseq 2000 platform, by Illumina (25 mentions)
Hiseq 4000 platform, by Illumina (23 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (22 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (20 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (14 mentions)
703 protocols using qubit rna assay kit
1
Extraction and Purification of Total RNA from Immune Cells
2
Transcriptome Analysis of Cannabis Hypocotyls
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Cannabis sativa (cv. Santhica 27) hypocotyls of 6, 9, 15, and 20 days after sowing were grown in controlled conditions in incubators following a cycle of 16 h light 25°C/8 h dark 20°C. Three biological replicates per time point were analyzed. Each biological replicate consisted of 20 hypocotyls randomly selected among all incubators. The pooling of hypocotyls was needed because of the quantity of material required for transcriptomics. In order to bring to a minimum this pooling bias, a high number of hypocotyls were pooled together. By doing so, the power to detect differentially expressed genes within the four populations increased (Rajkumar et al., 2015 (link)). Sampling was performed on a single experimental batch to minimize technical variability. Samples were immediately frozen in liquid nitrogen and conserved at -80°C until RNA extraction. The sampled hypocotyls were crushed to a fine powder using a mortar, a pestle and liquid nitrogen. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen), treated with DNase I on column, quantified and quality-checked using a Qubit 2.0 Fluorometer (Invitrogen) with the Qubit RNA Assay Kit (Molecular Probes), a NanoDrop 1000 Spectrophotometer (Thermo Scientific) and a 2100 Bioanalyzer (Agilent Life Sciences). All the RNAs displayed a RIN above 7.
3
RNA Isolation and cDNA Synthesis
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Blood samples from patients were collected and stored at -80 °C for RNA isolation. Total RNA was isolated by Purelink RNA Mini Kit (Invitrogen, Cat. 12183018A) according to the manufacturer's instructions. The concentration of total RNA was analyzed by Qubit RNA Assay Kit and Qubit 4 Fluorometer (Invitrogen/Molecular Probes). Then, 100 ng of total RNA was reverse transcribed with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).
4
RNA Extraction and Characterization for Spheroid Analysis
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Total RNAs with retention of small RNAs were extracted from the parental (as control groups) and spheroids (as experimental groups) using Exiqon miRCURY RNA Isolation Kit (Exiqon, Denmark) in line with the manufacturer's instructions. A total of six samples (three for parental and three for spheroids) were used. RNA was treated with DNase I (Qiagen, USA) to eliminate genomic DNA contamination. The concentration and purity of extracted RNA was determined by OD measurements of aliquots at a wavelength of 260/280 nm and further checked using Qubit RNA Assay Kit (GIBCO, USA). The integrity of the RNA samples was determined by Agilent 2100 Bioanalyzer using RNA 6000 Pico Kit (Agilent Technologies, USA). Only RNA samples with sufficient concentration (600 ng/µL) and RNA Integrity Number (RIN)>8 were used for libraries preparation for next generation sequencing library preparation and quantitative real time PCR (qRT-PCR) validation.
5
RNA Extraction and Quantification Protocol
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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), a trypsin–EDTA (ethylenediaminetetraacetic acid) solution (0.05% trypsin–EDTA), β-mercaptoethanol, TRIzol, and a Qubit® RNA Assay Kit were acquired from Gibco™ and Invitrogen™ (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Penicillin (10,000 units/mL)/streptomycin (10,000 μg/mL) antibiotics and trypsin were obtained from Merck-Millipore. Cell culture dishes were purchased from Corning, Inc. (Corning, NY, USA). The SYBR® Premix Ex Taq and PrimeScript TM 1st Strand complementary DNA (cDNA) Synthesis Kit were from TaKaRa Biotechnology (Dalian, China). M-MuLV Reverse Transcriptase (RNase H-), USER Enzyme, Phusion High-Fidelity DNA polymerase, and NEBNext® UltraTM RNA Library Prep Kit was from Illumina® (NEB, Ipswich, MA, USA).
6
RNA Isolation and Quality Assessment
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TransZol Up (Transgen, Beijing, China) was used to isolate total RNA from each sample (IR-CK, no BPH; IR56-BPH infested, IR-IR56-BPH; TN1-BPH infested, IR-TN1-BPH) (Figure 9 ). The isolation of total RNA was performed, as described by Nanda et al. [23 (link)]. Subsequently, the purity and concentration of total RNAs were detected using a NanoPhotometer spectrophotometer (Thermo-Fischer Scientific, Waltham, MA, USA). Then, the integrity of isolated RNA was evaluated using a Qubit RNA Assay Kit coupled with Qubit 2.0 Flurometer (Thermo-Fischer Scientific, Waltham, MA, USA) from Agilent 2100 (Thermo-Fischer Scientific, Waltham, USA). To ensure the application of qualified samples for sequencing, electrophoresis of 1% (w/v) agarose gels was used to monitor degradation and contamination of RNA.
7
RNA-Seq Protocol for Leukocytes
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A 20-mL blood sample was obtained from the caudal vein from each animal for buffy coat (leukocytes) collection using 15 min centrifugation at 3,000 r/min.
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from leukocytes according to the manufacturer’s protocol. The RNA quality was checked on a 1% agarose gel and quantified using a Qubit RNA Assay Kit and a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA USA). RNA integrity was assessed with the BioAnalyzer 2100 System (Agilent Technologies, Santa Clara, CA, USA). All 16 RNA samples had an RNA integrity number (RIN) larger than 7.0 (Supplementary Table S1 ). The 28S:18S rRNA ratios of all samples were larger than 1.7 (Supplementary Table S1 ). An equivalent amount (4 μg) of total PBL RNA purified from each animal was used to construct RNA-Seq libraries with the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, Ipswich, MA, USA). Finally, the libraries were sequenced using 150 bp paired-end reads with the Illumina HiSeq X Ten System (CapitalBio Technology, Beijing, China).
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from leukocytes according to the manufacturer’s protocol. The RNA quality was checked on a 1% agarose gel and quantified using a Qubit RNA Assay Kit and a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA USA). RNA integrity was assessed with the BioAnalyzer 2100 System (Agilent Technologies, Santa Clara, CA, USA). All 16 RNA samples had an RNA integrity number (RIN) larger than 7.0 (Supplementary Table S
8
RNA Extraction and Circular RNA Analysis
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RNA degradation and contamination was monitored on 1% agarose gels. According to the manufacturer's protocol, each tissue sample was washed three times using cold PBS and 1 ml TRIzol® reagent was added (Thermo Fisher Scientific, Inc.) to extract the RNA. The RNA concentration was measured using the Qubit® RNA assay kit in a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Inc.). RNA integrity was verified using an RNA Nano 1000 assay kit for the Bioanalyzer 2100 system (Agilent Technologies, Inc.). The method for determining the levels of lncRNAs and miRNAs was the same as that used for mRNAs. For the quantification of circRNAs, exonuclease was used to exclude non-circRNAs. The RNA was divided into two copies. Linear RNA was digested with RNase R (cat. no. RNR07250; Epicentre; Illumina, Inc.) to leave only the circRNAs. The other half of the sample from the same RNA extraction was not treated with RNase R. The two samples of RNA were reverse transcribed according to a previous study (25 (link)). The sample treated with RNase R was used to examine the expression of circRNAs and the other sample that was not treated with RNase R was used to measure the expression of β-actin.
9
Comprehensive RNA Extraction and Analysis
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Total RNA was extracted from the samples using TRIzol® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. RNA degradation and contamination were monitored using 1% agarose gels. RNA purity was examined using a NanoPhotometer® spectrophotometer (Implen, Inc., Westlake Village, CA, USA). The RNA concentration was measured using the Qubit® RNA Assay kit in a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Inc.). RNA integrity was assessed using the RNA Nano 6000 Assay kit for the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).
10
Nanostring Profiling of T Cell Activation
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The nCounter Nanostring GX Human Immunology V2 panel (Nanostring, Seattle, WA) was used to analyze the expression of 579 immune and inflammation-associated target genes and 15 reference control genes in naive CD4+ T cells that were cultured for 4 h with either untreated or IFNγ-treated keratinocytes. Before RNA isolation, the T cells were isolated using flow cytometry. Total RNA was then extracted from 1 × 106 naive T cells using the Direct-zol RNA Miniprep Kit (Zymo Research, Freiburg). All RNA samples were quantified by using a Qubit RNA assay kit (Thermo Fisher Scientific, Waltham, MA), and RNA integrity was assessed using an Agilent 2100 Bioanalyzer system.
A total of 25 µg total RNA (5 µL/sample) was mixed with nCounter® reporter CodeSet (3 µL) and nCounter® capture ProbeSet (2 µL) with a hybridization buffer (5 µL) for an overnight hybridization reaction at 65 °C. The reaction was cooled to 4 °C, the samples were purified and immobilized on a cartridge, and the data were assessed on the nCounter SPRINT Profiler. The exported data were analyzed using Nanostring nSolver 4.0. A detailed description of the data analysis is described in the supplementary information.
A total of 25 µg total RNA (5 µL/sample) was mixed with nCounter® reporter CodeSet (3 µL) and nCounter® capture ProbeSet (2 µL) with a hybridization buffer (5 µL) for an overnight hybridization reaction at 65 °C. The reaction was cooled to 4 °C, the samples were purified and immobilized on a cartridge, and the data were assessed on the nCounter SPRINT Profiler. The exported data were analyzed using Nanostring nSolver 4.0. A detailed description of the data analysis is described in the supplementary information.
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