Quant it rna br assay kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Quant-iT RNA BR Assay Kit is a fluorescence-based reagent designed for the quantification of RNA samples. It provides a sensitive and accurate method to measure RNA concentration in solution. The kit includes all the necessary components to perform the assay, including the RNA-binding reagent, standard RNA samples, and buffers.

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Lab products found in correlation

Qubit 3.0 fluorimeter, by Thermo Fisher Scientific (4 mentions) Experion rna highsens analysis kit, by Bio-Rad (3 mentions) Experion instrument, by Bio-Rad (3 mentions) Rnaeasy mini kit, by Qiagen (3 mentions) Rneasy mini kit, by Qiagen (3 mentions) Quick rna miniprep kit, by Zymo Research (3 mentions) Qubit fluorimeter, by Thermo Fisher Scientific (2 mentions) Experion automated electrophoresis system, by Bio-Rad (2 mentions) Zr whole blood total rna kit, by Zymo Research (2 mentions) Rnalater buffer, by Thermo Fisher Scientific (1 mentions) Direct zol rna miniprep kit, by Zymo Research (1 mentions) Bioanalyzer, by Agilent Technologies (1 mentions) Qubit fluorometer, by Thermo Fisher Scientific (1 mentions) Hiseq 2500 sequencer, by Illumina (1 mentions) Truseq exome enrichment kit, by Illumina (1 mentions) Axyprep blood genomic dna miniprep kit, by Corning (1 mentions) The agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions) Revertra ace qpcr rt master mix with gdna remover, by Toyobo (1 mentions) Sv total rna isolation system, by Promega (1 mentions) Malachite green, by Merck Group (1 mentions)

12 protocols using quant it rna br assay kit

Isolation of Total RNA from Brain Tissue

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During isolating RNA, tissue samples from each animal were homogenized using TRI Reagent (MRC, USA) [35 ]. Further phase separation was performed with addition of chloroform to the brain tissue homogenate with TRI Reagent according to the manufacturer's recommendations. After phase separation, the aqueous phase containing RNA was taken and then ethanol was introduced in a 1: 1 ratio. Further, the isolation of total RNA was performed using RNAeasy Mini Kit (Qiagen, Germany) according to the manufacturer's recommendations. RNA concentration was measured using a Quant-iT RNA BR Assay Kit and a Qubit 3.0 fluorimeter (Invitrogen, USA).
RNA quality was assessed using the Experion RNA HighSens Analysis Kit and the Experion instrument (Bio-Rad, USA).

Rudenok M.M., Alieva A.K., Starovatykh J.S., Nesterov M.S., Stanishevskaya V.A., Kolacheva A.A., Ugryumov M.V., Slominsky P.A, & Shadrina M.I. (2020). Expression analysis of genes involved in mitochondrial biogenesis in mice with MPTP-induced model of Parkinson's disease. Molecular Genetics and Metabolism Reports, 23, 100584.

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Transcriptomic Analysis of Rat Hippocampus

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The hippocampus and prefrontal cortex were dissected from the whole brain. The dissection took no more than 10 min per animal. Samples were flash frozen, homogenized a using rotor homogenizer, and then soaked in RNA-later buffer (Thermo Fisher Scientific).
Total RNA was isolated from a 10-mg sample of tissue from the frontal cortex (F) and hippocampus (H) of the brains of rats. Total RNA isolation was performed from the rat brains for expression analysis of individual genes for each individual animal. Total RNA isolation was conducted using a Direct-Zol RNA Mini-Prep Kit (Zymo Research) according to the manufacturer's recommendations. The concentration of isolated total RNA was measured using a Quant-iT RNA BR Assay Kit and a Qubit fluorimeter (Invitrogen). RNA quality was monitored using an Experion automated electrophoresis system (Bio-Rad Laboratories). The RNA quality index, measured using BioAnalyzer (Agilent), was higher than 8.5 in all samples.
Whole-transcriptome analysis was performed using samples of tissue isolated from hippocampus of the rats brain. For this purpose, 5 mg of brain tissue were taken from each of the 8 animals in each group (the control group, the group decapitated 20 min after the FST, and the group decapitated 24 h after the FST).

Vlasov I., Filatova E., Slominsky P, & Shadrina M. (2023). Differential expression of Dusp1 and immediate early response genes in the hippocampus of rats, subjected to forced swim test. Scientific Reports, 13, 9985.

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RNA Extraction and Normalization from Beetle

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RNA was extracted from 23 adults and 142 larvae of L. decemlineata using the RNeasy Mini Kit (Qiagen) following the manufacturer's protocol for animal tissues. The RNA was extracted from 20 mg of tissue from the head, thorax or abdomen of adult beetles, from the head of 3^rd and 4^th instar larvae and from pools of entire 1^st and 2^nd instar larvae. Extracted RNA was checked for integrity and size and then quantified using a Quant-IT RNA BR assay kit (Invitrogen). The single extracts were diluted to obtain a concentration of 100 ng/μl. Extraction was conducted in two different laboratories, and two pools of equal amounts of RNA were obtained, one for Russia and Camposampiero and one for Montello, for both adults and larvae. Each pool, consisting of a total of 5 µg of RNA, was stored in pure ethanol and shipped to Evrogen Labs Ltd., Moscow. The two adult and the two larval RNA pools were used for ds cDNA synthesis using the SMART approach [87] (link). SMART-prepared amplified cDNAs were pooled (ratio: ¾ of the Russia/Camposampiero pool and ¼ of the Montello pool) and then normalized using the DSN normalization method [88] (link) to reduce overabundant transcripts. Normalization included cDNA denaturation/reassociation, a treatment by duplex-specific nuclease (DSN [89] (link)) and the amplification of the normalized fraction by PCR.

Kumar A., Congiu L., Lindström L., Piiroinen S., Vidotto M, & Grapputo A. (2014). Sequencing, De Novo Assembly and Annotation of the Colorado Potato Beetle, Leptinotarsa decemlineata, Transcriptome. PLoS ONE, 9(1), e86012.

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Isolation and Quantification of Total RNA

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Tissue samples from each animal were homogenized using TRI Reagent (MRC, USA). Further fractionation was carried out with the introduction of chloroform to the brain tissue homogenate in TRI Reagent according to the manufacturer’s recommendations. After fractionation, the aqueous phase containing RNA was taken and ethanol was introduced in a 1:1 ratio. Phenol–chloroform fractionation was used to isolate the protein (see below).
Further isolation of total RNA was carried out using RNeasy Mini Kit (Qiagen, Germany) and Quick-RNA MiniPrep Kit (Zymo Research Corp., USA) according to the manufacturer’s recommendations. RNA concentration was measured using Quant-iT RNA BR Assay Kit and a Qubit 3.0 fluorimeter (Invitrogen, USA). RNA quality was assessed using the Experion RNA HighSens Analysis Kit and the Experion instrument (Bio-Rad, USA).

Alieva A., Rudenok M., Filatova E., Karabanov A., Doronina O., Doronina K., Kolacheva A., Ugrumov M., Illarioshkin S., Slominsky P, & Shadrina M. (2020). VCP expression decrease as a biomarker of preclinical and early clinical stages of Parkinson’s disease. Scientific Reports, 10, 827.

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RNA Extraction from Brain Tissue

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Tissue samples from each animal were homogenized using TRI Reagent (MRC, Cincinnati, OH, USA; Molecular Research Center, 2017). Further phase separation was performed with the addition of chloroform to the brain tissue homogenate with TRI Reagent according to the manufacturer’s recommendations. After phase separation, the aqueous phase containing RNA was separated and mixed with ethanol in a 1:1 ratio. The phenol-chloroform fraction was used for protein isolation (see below). Further isolation of total RNA was performed using RNAeasy Mini Kit (Qiagen, Solingen, Germany) and Quick-RNA Miniprep Kit™ (Zymo Research Corp., Irvine, CA, USA) according to the manufacturer’s recommendations. The RNA concentration was measured using a QuantiT RNA BR Assay Kit and a Qubit 3.0 fluorimeter (Invitrogen, Carlsbad, CA, USA). RNA quality was assessed using the Experion RNA HighSens Analysis Kit and the Experion instrument (Bio-Rad, Hercules, CA, USA).

Sarkisova K.Y., Fedosova E.A., Shatskova A.B., Rudenok M.M., Stanishevskaya V.A, & Slominsky P.A. (2023). Maternal Methyl-Enriched Diet Increases DNMT1, HCN1, and TH Gene Expression and Suppresses Absence Seizures and Comorbid Depression in Offspring of WAG/Rij Rats. Diagnostics, 13(3), 398.

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Whole Exome Sequencing for Variant Identification

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Genomic DNA was obtained from leukocytes using AxyPrepBlood Genomic DNA Miniprep Kit (“Axygen”, USA) as recommended by the manufacturer. The concentration of isolated nucleic acids was measured using Qubit fluorometer (“Invitrogen”, USA) and Quant-iT RNA BR Assay Kit commercial kit as recommended by the manufacturer.
WES was performed using a TruSeq Exome Enrichment kit (Illumina, San Diego, CA, USA) on an Illumina HiSeq 2500 sequencer. Sequence consisted of 201121 exons in 20794 genes with an average coverage of 40×, providing genotype data for 90% of consensus coding exon bases. Resulting potentially pathogenically significant variants in genes listed in Supplementary Table S1 were validated by Sanger sequencing.

Shulskaya M.V., Alieva A.K., Vlasov I.N., Zyrin V.V., Fedotova E.Y., Abramycheva N.Y., Usenko T.S., Yakimovsky A.F., Emelyanov A.K., Pchelina S.N., Illarioshkin S.N., Slominsky P.A, & Shadrina M.I. (2018). Whole-Exome Sequencing in Searching for New Variants Associated With the Development of Parkinson’s Disease. Frontiers in Aging Neuroscience, 10, 136.

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Whole Blood RNA Isolation Protocol

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Blood samples were collected from subjects at 8 a.m. before eating and stored at +4°C for no more than 2 h before RNA isolation. Total RNA was isolated from peripheral blood using a ZR Whole-Blood Total RNA Kit (〈〈Zymo Research Corp.〉〉, USA). RNA quantities were determined by spectrophotometry using a Quant-iT RNA BR Assay Kit and a Qubit fluorimeter (Invitrogen, USA). RIN values were measured by using The Agilent 2100 Bioanalyzer system (Agilent Technologies, USA) and were above 8.

Alieva A.K., Filatova E.V., Karabanov A.V., Illarioshkin S.N., Slominsky P.A, & Shadrina M.I. (2015). Potential Biomarkers of the Earliest Clinical Stages of Parkinson's Disease. Parkinson's Disease, 2015, 294396.

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HepG2 Cell Culture and RNA Isolation

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HepG2 cells were seeded at a density of 5.0 × 10⁴ cells/cm² in six-well plates and cultured in DMEM with 10% FBS for 3, 6 and 12 days. Cells were harvested and pelleted followed by total RNA isolation using an SV Total RNA Isolation System (Promega, Madison, WI). The concentration of total RNA was measured using a Quant-iT RNA BR Assay Kit (Invitrogen, Carlsbad, CA, USA). To obtain cDNA, 0.5 μg of total RNA was used as the template for reverse transcription using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. cDNA was diluted 1:20 in nuclease-free water before being used as a template for qPCR.

Tsuji T., Morita S.Y., Nakamura Y., Ikeda Y., Kambe T, & Terada T. (2021). Alterations in cellular and organellar phospholipid compositions of HepG2 cells during cell growth. Scientific Reports, 11, 2731.

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Molecular Techniques for Cell Analysis

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DMEM/F-12 growth medium, Glutamax, Hu IL-8 Cytoset ELISA kit, PureLink Genomic DNA mini kit, Quant-iT RNA BR assay kit, NuPAGE Novex 4–12% Bis-Tris gel, TRIzol reagent, DNAse1-RNAse-free (AM2222), Superscript III reverse transcriptase, RNAseOUT recombinant Ribonuclease inhibitor, dNTP, DTT, aprotinin, Lipofectamine, microAMp optical 384 well reaction plate, custom-made primers, and 1 kb plus DNA ladder were purchased from Life Technologies (Burlington, ON, Canada). Normocin was obtained from InvivoGen (San Diego, CA, USA). ATP, ADP, AMP, adenosine, ATP-γ-S, suramin, diadenosine pentaphosphate (Ap₅A), malachite green, and random nonamers (R7647) were purchased from Sigma. Millex GP syringe-driven filter unit 0.22 µm and Immobilon-P membrane were from Millipore (Billerine, MA, USA). Hank's balanced salt solution (HBSS) with Ca²⁺ and Mg²⁺, Hepes, and antibiotic-antimycotic solutions was from Wisent (St. Bruno, QC, Canada). RNeasy mini kit and QuantiTect Reverse Transcription kit were from Qiagen (Mississauga, ON, Canada). Taq polymerase was obtained from New England Biolabs (Ipswich, MA, USA) and FastStart Universal SYBR Green Master (Rox) was from Roche (Mannheim, Germany).

Bahrami F., Kukulski F., Lecka J., Tremblay A., Pelletier J., Rockenbach L, & Sévigny J. (2014). Purine-Metabolizing Ectoenzymes Control IL-8 Production in Human Colon HT-29 Cells. Mediators of Inflammation, 2014, 879895.

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RNA Extraction from Brain and Blood

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The isolation of total RNA from brain tissues was carried out using the RNAeasy Mini Kit (Qiagen, Germany) and Quick-RNA MiniPrep Kit (Zymo Research Corp., Irvine, CA, USA).
The isolation of total RNA from 250 µL of each sample of human and mouse peripheral blood was performed using Whole Blood RNA MiniPrep Kit (Zymo Research Corp., Irvine, CA, USA).
The concentration of total isolated RNA from all samples was measured using a Quant-iT RNA BR Assay Kit and a Qubit 3.0 fluorimeter (Life Technologies, Grand Island, NY, USA). RNA quality was monitored using an Experion automated electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA). The RNA quality index was higher than 8.5 in all samples.
All procedures were carried out according to the manufacturers’ recommendations.

Alieva A.K., Filatova E.V., Rudenok M.M., Slominsky P.A, & Shadrina M.I. (2021). Housekeeping Genes for Parkinson’s Disease in Humans and Mice. Cells, 10(9), 2252.

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