Qubit dsdna hs assay

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, Canada, United Kingdom

The Qubit dsDNA HS Assay is a quantitative fluorescence-based method for measuring the concentration of double-stranded DNA (dsDNA) samples. It provides accurate measurements of dsDNA concentrations from 0.2 ng/mL to 100 ng/mL.

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Lab products found in correlation

Qubit 2.0 fluorometer, by Thermo Fisher Scientific (84 mentions) Qubit fluorometer, by Thermo Fisher Scientific (38 mentions) Qubit 3.0 fluorometer, by Thermo Fisher Scientific (32 mentions) Nextseq 500, by Illumina (31 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (22 mentions) Ampure xp bead, by Beckman Coulter (21 mentions) 2100 bioanalyzer, by Agilent Technologies (18 mentions) Bioanalyzer, by Agilent Technologies (18 mentions) Hiseq 2500, by Illumina (16 mentions) Tapestation 4200, by Agilent Technologies (15 mentions) Qubit 4 fluorometer, by Thermo Fisher Scientific (13 mentions) Dneasy blood and tissue kit, by Qiagen (13 mentions) Qiaamp dna mini kit, by Qiagen (13 mentions) Nanodrop, by Thermo Fisher Scientific (12 mentions) Qiaamp circulating nucleic acid kit, by Qiagen (12 mentions) Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (12 mentions) Agencourt ampure xp bead, by Beckman Coulter (11 mentions) Dneasy powersoil kit, by Qiagen (11 mentions) Miseq system, by Illumina (11 mentions) Novaseq 6000, by Illumina (10 mentions)

Close spelling variants of this product

Qubit assay (159 citations) Qubit dsDNA assay (43 citations) Qubit dsDNA HS (29 citations) DsDNA HS assay (25 citations) DsDNA Qubit (3 citations)

338 protocols using qubit dsdna hs assay

Tumor Mutational Burden Profiling

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40 ng DNA quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) was used for library preparation with the QIAseq Human Tumor Mutational Burden Panel (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Final libraries were quantified Qubit dsDNA HS Assay (Thermo Fisher Scientific), pooled and sequenced on a NextSeq 500 (Illumina).
Quality of the NextSeq 500 (Illumina) sequencing runs were assessed with the Illumina Sequencing Analysis Viewer (Illumina). Sequencing data was analyzed with ‘Identify QIAseq DNA Somatic Variants with TMB Score (Illumina)’ v1.47 in the plugin ‘Biomedical Genomics Analysis v 1.2′ on the CLC Genomics Workbench v12.0.2 (Qiagen).
In addition to the Qiagen software, we also analyzed the data with our in-house pipeline (see description above) with minor modifications regarding the extraction of the umi (unique molecular index). Due to the different chemistry for library preparation, we also sequenced 15 normal samples independent from tumors that served as a panel of normal.
Variant annotation for filtering was done with Mutect2 FilterMutectCalls. Read_position and strand_artifact filter flags were removed for subsequent analysis. Further we employed the LearnReadOrientationModel of GATK to filter strand biases.

Heydt C., Rehker J., Pappesch R., Buhl T., Ball M., Siebolts U., Haak A., Lohneis P., Büttner R., Hillmer A.M, & Merkelbach-Bruse S. (2020). Analysis of tumor mutational burden: correlation of five large gene panels with whole exome sequencing. Scientific Reports, 10, 11387.

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Transcriptome analysis of NP swab and whole blood samples

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Extracted RNA from NP swab samples (25 μl) was treated with a nuclease cocktail of TURBO DNase (Thermo Fisher Scientific) and Baseline Zero DNase (Ambion) for 30 min at 37°C and purified using AMPure XP beads (Beckman Coulter) on the epMotion 5075 (Eppendorf). Purified RNA (7 μl) was used for library preparation using the SMART-Seq Stranded kit (Takara Bio) and purified using AMPure XP beads (Beckman Coulter) on the epMotion 5073 (Eppendorf). Libraries were quantified using the Qubit dsDNA HS Assay (Thermo Fisher Scientific) on the Qubit Flex (Thermo Fisher Scientific).
WB sample libraries were prepared using 9 μl of total RNA and TruSeq Total RNA with Ribo-Zero Globin (Illumina) and spiked with 1 μl of ERCC RNA Spike-In Mix (Thermo Fisher Scientific). Libraries were purified using AMPure XP beads (Beckman Coulter) and quantified using the Qubit dsDNA HS Assay (Thermo Fisher Scientific) on the Qubit Flex (Thermo Fisher Scientific).
NP swab and WB sample libraries were sequenced on the NovaSeq 6000 (Illumina) using 150–base pair paired-end sequencing at the UCSF Center for Advanced Technology. Included in each sequencing run were negative controls (nuclease-free water) to monitor for laboratory and reagent contamination and a Human Reference RNA Standard (Agilent) to monitor for sequencing efficiency.

Ng D.L., Granados A.C., Santos Y.A., Servellita V., Goldgof G.M., Meydan C., Sotomayor-Gonzalez A., Levine A.G., Balcerek J., Han L.M., Akagi N., Truong K., Neumann N.M., Nguyen D.N., Bapat S.P., Cheng J., Martin C.S., Federman S., Foox J., Gopez A., Li T., Chan R., Chu C.S., Wabl C.A., Gliwa A.S., Reyes K., Pan C.Y., Guevara H., Wadford D., Miller S., Mason C.E, & Chiu C.Y. (2021). A diagnostic host response biosignature for COVID-19 from RNA profiling of nasal swabs and blood. Science Advances, 7(6), eabe5984.

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DNA Extraction from Tumor and Blood Samples

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Fresh-frozen tumor DNA was extracted using DNeasy Blood and Tissue Kits (69506, Qiagen Ltd), according to the manufacturer’s instructions. Blood DNA was extracted from whole blood using Flexigene DNA Kits (51206, Qiagen Ltd). All samples were quantified using a NanoDrop (ND1000; Thermoscientific) and Qubit® dsDNA HS Assay (Q32851; Life Technologies) and DNA size and quality were tested using gel electrophoresis. Samples with a concentration of less than 50 ng/µl, or absence of a high molecular weight band in electrophoresis gels, were excluded from further analyses.
DNA from tumor samples obtained as FFPE tissue cores was extracted with QIAamp FFPE Tissue kits (Qiagen), according to the manufacturer’s instructions and quantified using the Qubit® dsDNA HS Assay (Q32851; Life Technologies)⁴¹.

Newell F., Kong Y., Wilmott J.S., Johansson P.A., Ferguson P.M., Cui C., Li Z., Kazakoff S.H., Burke H., Dodds T.J., Patch A.M., Nones K., Tembe V., Shang P., van der Weyden L., Wong K., Holmes O., Lo S., Leonard C., Wood S., Xu Q., Rawson R.V., Mukhopadhyay P., Dummer R., Levesque M.P., Jönsson G., Wang X., Yeh I., Wu H., Joseph N., Bastian B.C., Long G.V., Spillane A.J., Shannon K.F., Thompson J.F., Saw R.P., Adams D.J., Si L., Pearson J.V., Hayward N.K., Waddell N., Mann G.J., Guo J, & Scolyer R.A. (2019). Whole-genome landscape of mucosal melanoma reveals diverse drivers and therapeutic targets. Nature Communications, 10, 3163.

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Exome Sequencing of Blood DNA

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Genomic DNA was isolated from peripheral blood samples of the probands and their parents using the TIANamp Blood DNA Kit (TIANGEN, Beijing, China) following the manufacturer’s instructions and used for library preparation. The exon regions along with adjacent intron regions (±20 bp) of all genes contained in post-PCR libraries were then captured and enriched. Enriched sample libraries were quantified by Qubit dsDNA HS Assay (Thermal Fisher Scientific), and library size was checked by High Sensitivity D1000 Reagents (Agilent, United States). Final libraries were sequenced using the Human Exome Probes P039-Exome (MyGenostics, Beijing, China) on an Illumina NovaSeq 5000 sequencer (Illumina, United States).

Gao M., Yu F., Dong R., Zhang K., Lv Y., Ma J., Wang D., Zhang H., Gai Z, & Liu Y. (2023). Diagnostic application of exome sequencing in Chinese children with suspected inherited kidney diseases. Frontiers in Genetics, 13, 933636.

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Soil Microbiome Analysis Protocol

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DNA was extracted form 0.25 g freeze‐dried rhizosphere, soil and root samples by DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA concentration was assessed by Qubit dsDNA HS Assay (Termo Fisher Scientific, Waltham, Massachusetts). The DNA was used for quantifying ITS2 and 16S rRNA copy numbers (qPCR). ¹³C‐enriched DNA was obtained from rhizosphere samples by CsCl gradient fractionation. This, together with soil, rhizosphere and root DNA, was subjected to fungal and bacterial amplicon sequencing. For details on molecular analyses, see the Supporting Information.

Clocchiatti A., Hannula S.E., Hundscheid M.P., klein Gunnewiek P.J, & de Boer W. (2021). Stimulated saprotrophic fungi in arable soil extend their activity to the rhizosphere and root microbiomes of crop seedlings. Environmental Microbiology, 23(10), 6056-6073.

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Mouse Cortex DNA Extraction and RRBS Sequencing

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Genomic DNA was isolated from mouse cortex [44 (link)] using the AllPrep DNA/RNA Mini Kit (QIAGEN) and assessed for quality and quantity using the NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific) and the Qubit high sensitivity assay (Qubit dsDNA HS Assay, Thermo Fisher Scientific). RRBS libraries were prepared using the Premium RRBS kit (Diagenode). The final RRBS library pools were distributed across thirty-two HiSeq2500 (Illumina) lanes and subjected to 50 bp single-end sequencing as previously described [20 (link)].

Seiler Vellame D., Castanho I., Dahir A., Mill J, & Hannon E. (2021). Characterizing the properties of bisulfite sequencing data: maximizing power and sensitivity to identify between-group differences in DNA methylation. BMC Genomics, 22, 446.

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DNA Extraction from Mammalian and Yeast Cells

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MRC-5 cells were pre-treated with 1 mg/ml collagenase prior to trypsinization. Cell were pelled (MRC-5 after trypsin neutralization, HeLa S3—after FACS) and resuspended in 10 mM Tris–HCl pH 8.0, 0.5% (w/v) SDS, 100 mM EDTA supplemented with 20 μg/ml RNase A and 1 mg/ml proteinase K and incubated at 50°C for at least 1 h.
After the RNase A and proteinase K treatment, DNA samples from yeast and mammalian cells were allowed to cool to room temperature and sample volumes were adjusted to 0.5 ml by addition of TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). The samples were mixed with an equal volume of phenol:chloroform:isoamyl alcohol pH 8.0 (25:24:1; Sigma) followed by single or double extraction using an equal volume of chloroform. DNA was then precipitated using isopropanol in the presence of potassium acetate. DNA concentration was measured using the Qubit™ dsDNA HS assay (ThermoFisher).

Batrakou D.G., Heron E.D, & Nieduszynski C.A. (2018). Rapid high-resolution measurement of DNA replication timing by droplet digital PCR. Nucleic Acids Research, 46(19), e112.

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DNA Amplification and Sequencing Workflow

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After reactions were terminated through heat, we used isobutanol to demulsify the water-in-oil droplets and used Zymo-Spin™ columns (Zymo Research) coupling with DNA Clean & Concentrator kit (Zymo Research) kit to purify the amplified DNA following the recommended protocol. Quality control of the amplification result was performed by measuring DNA amount using Qubit dsDNA HS Assay (Thermo Fisher Scientific) and evaluating the amplification bias through quantitate PCR (Supplementary Fig. 3, Supplementary Table 1). Sequencing libraries were built for Illumina platform using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme) using 50 ng DNA as input. The libraries were sequenced on Illumina Hiseq 4000 or Hiseq 2500 platform. The sequencing details of each library are listed at Supplementary Table 2.

Fu Y., Zhang F., Zhang X., Yin J., Du M., Jiang M., Liu L., Li J., Huang Y, & Wang J. (2019). High-throughput single-cell whole-genome amplification through centrifugal emulsification and eMDA. Communications Biology, 2, 147.

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Metagenomic Next-Generation Sequencing Protocol

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Metagenomic next-generation sequencing (mNGS) was performed as previously described [17 ,56 (link)]. Briefly, 18 μL of extracted RNA was treated with Turbo DNAse (ThermoFisher, Waltham, MA). First strand cDNA synthesis was completed using SuperScript IV (ThermoFisher) and random hexamers (Invitrogen, Carlsbad, CA) followed by second strand synthesis by Sequenase version 2.0 (ThermoFisher). The resulting cDNA was purified using either the DNA Clean & Concentrator kit (Zymo, Irvine, California) or 1.6× volumes of AMPure XP beads (Beckman Coulter, Brea, CA). Library preparation was performed using the Nextera XT Kit (Illumina, San Diego, CA). Libraries were cleaned with 0.7× or 0.75× volumes of Ampure beads (Beckman Coulter), quantified using either the Qubit dsDNA HS assay (ThermoFisher) or Quant-iT dsDNA HS assay (ThermoFisher), quality checked by Bioanalyzer or TapeStation (Agilent, Santa Clara, CA), pooled, and sequenced on 1 × 75 bp runs on an Illumina NextSeq or 1 × 101 bp runs on an Illumina NovaSeq.

Lieberman N.A., Peddu V., Xie H., Shrestha L., Huang M.L., Mears M.C., Cajimat M.N., Bente D.A., Shi P.Y., Bovier F., Roychoudhury P., Jerome K.R., Moscona A., Porotto M, & Greninger A.L. (2020). In vivo antiviral host transcriptional response to SARS-CoV-2 by viral load, sex, and age. PLoS Biology, 18(9), e3000849.

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Microbiome Analysis of Colon Lumen and Mucosa

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To analyze the microbiome of colon lumen contents, 2–3 colon pellets from each mouse were collected and frozen at −20 °C until use. To collect colonic mucosal scrapings, colons were longitudinally opened in a sterile dish and scraped with 1 ml PBS. Bacterial genomic DNA from frozen stool samples was extracted using QIAamp DNA Stool Mini Kit (QIAGEN). Purified DNA was quantified by Qubit dsDNA HS Assay (Thermo Fisher Cat# Q32854) and normalized. Amplicons were purified and quantified by Qubit dsDNA HS Assay and combined with equal mass to make a pooled library. The pooled library was multiplexed sequenced (Illumina MiSeq, 251 nt × 2 pair-end reads with 12 nt index reads) through the Harvard Biopolymer’s Facility. Raw sequencing data were processed with QIIME2. In brief, raw sequencing data were imported to QIIME2 and demultiplexed, then DADA2 was used for sequence quality control and feature table construction. The feature table was used for beta diversity analysis, taxonomic analysis, and differential abundance testing using QIIME2. Beta group significance was determined by permutational analysis of variance (PERMANOVA). Identification of taxa associated with different groups was determined using Analysis of Composition of Microbiomes (ANCOM).

Yang D., Jacobson A., Meerschaert K.A., Sifakis J.J., Wu M., Chen X., Yang T., Zhou Y., Anekal P.V., Rucker R.A., Sharma D., Sontheimer-Phelps A., Wu G.S., Deng L., Anderson M.D., Choi S., Neel D., Lee N., Kasper D.L., Jabri B., Huh J.R., Johansson M., Thiagarajah J.R., Riesenfeld S.J, & Chiu I.M. (2022). Nociceptor neurons direct goblet cells via a CGRP-RAMP1 axis to drive mucus production and gut barrier protection. Cell, 185(22), 4190-4205.e25.

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