Qubit 4 fluorometer

Manufactured by Agilent Technologies

Sourced in United States

The Qubit 4 Fluorometer is a compact, benchtop instrument designed for accurate and sensitive quantification of nucleic acids and proteins. It utilizes fluorescence-based detection methods to determine sample concentrations. The device provides rapid and reliable measurements, making it a versatile tool for various laboratory applications.

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Qubit fluorometer (113 citations) Qubit 4 (5 citations) Qubit 4.0 fluorometer (4 citations)

14 protocols using qubit 4 fluorometer

Shearing and Library Preparation for Illumina Sequencing

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After SPRI bead purification of the crosslink-reversed samples, we transferred DNA from each to Covaris® microTUBE AFA Fiber Snap-Cap tubes (Covaris Cat. No. 520045) and sonicated to an average length of 400 ±85 bp using a Covaris® ME220 Focused-Ultrasonicator™. Temperature was held stably at 6°C and treatment lasted sixty-five seconds per sample with a peak power of fifty watts, ten percent duty factor, and two-hundred cycles per burst. The average fragment length and distribution of sheared DNA was determined by capillary electrophoresis using an Agilent® FragmentAnalyzer 5200 and HS NGS Fragment Kit (Agilent Cat. No. DNF-474–0500). We ran sheared DNA samples twice through the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (Catalog No. E7645S) End Preparation and Adaptor Ligation steps with custom Y-adaptors to produce library preparation replicates. We purified ligation products via SPRI beads before Biotin enrichment using Dynabeads® MyOne™ Streptavidin C1 beads (ThermoFisher Catalog No. 65002).
We performed indexing PCR on streptavidin beads using KAPA HiFi HotStart ReadyMix (Catalog No. KK2602) and PCR products were isolated by SPRI bead purification. We quantified the libraries by Qubit™ 4 fluorometer and FragmentAnalyzer 5200 HS NGS Fragment Kit (Agilent Cat. No. DNF-474–0500) before pooling for sequencing on an Illumina HiSeq X at Fulgent Genetics.

Shafin K., Pesout T., Lorig-Roach R., Haukness M., Olsen H.E., Bosworth C., Armstrong J., Tigyi K., Maurer N., Koren S., Sedlazeck F.J., Marschall T., Mayes S., Costa V., Zook J.M., Liu K.J., Kilburn D., Sorensen M., Munson K.M., Vollger M.R., Monlong J., Garrison E., Eichler E.E., Salama S., Haussler D., Green R.E., Akeson M., Phillippy A., Miga K.H., Carnevali P., Jain M, & Paten B. (2020). Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomes. Nature Biotechnology, 38(9), 1044-1053.

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Comprehensive RNA Sequencing of Plant Flower Tissue

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Total RNA was extracted from the flower tissue of individual replicates using the Qiagen RNeasy Plus mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions after grinding in liquid nitrogen using a mortar and pestle. The RNA quality and quantity was assessed using the Nanodrop 8000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA), Qubit 4 Fluorometer (Waltham, MA, USA), and Agilent bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Sequencing library preparation and sequencing were performed by the University of Florida's Interdisciplinary Center for Biotechnology Research (ICBR, Gainesville, FL, USA). Equal parts of all 18 samples were pooled and submitted for two 8 M SMRT cells of PacBio IsoSeq sequencing on a Sequel II PacBio sequencer following the manufacturer's protocols (PacBio, Menlo Park, CA, USA). All 18 samples were also submitted individually for 8 GB of Illumina paired-end (150 bp) sequencing on a NovaSeq 6000 sequencer (Illumina, San Diego, CA, USA).

Parrish S.B., Paudel D, & Deng Z. (2023). Transcriptome analysis of Lantana camara flower petals reveals candidate anthocyanin biosynthesis genes mediating red flower color development. G3: Genes|Genomes|Genetics, 14(1), jkad259.

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Deep-Sea Microorganism Genome Sequencing

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The samples were deep-sea microorganisms provided by the Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, and their genomes were extracted by our colleagues. The V3–V4 region was cloned from the extracted genome using the following PCR mix solution: 2× Phanta Max Master Mix, 0.4 µM forward and reverse primers, and 150 ng DNA. The thermocycling process consisted of the following steps: initial denaturation at 95 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 15 s, annealing at 57 °C for 15 s, and extension at 72 °C for 30 s. At 72 °C, a 5 min extension step was then carried out. After that, the amplicons were processed for library preparation using the kits ND607, N616 and N805, and the product was characterized by the Qubit™ 4 Fluorometer and the Agilent 2100 Bioanalyzer.

Huang J., Li H., Shu F., Zhou W., Wu Y., Wang Y., Lv X., Gao M., Song Z, & Zhao S. (2024). Low-Temperature and High-Efficiency Solid-Phase Amplification Based on Formamide. Micromachines, 15(5), 565.

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16S DNA Library Preparation with Optimized Cleanup

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16S library preparation for DNAs from all groups was performed per our published protocol.23 (link) An optimised clean-up step was performed with Agencourt AMPure XP beads using modifications proposed by Quail et al,24 (link) and 5 µL of the final Amplicon PCR product was used for the Index PCR, which was performed using the Nextera XT Index kit and 2 x KAPA HiFi HotStart Ready Mix, following the Illumina protocol. After a second modified clean-up step, the final product was quantified using Qubit ds DNA BR Assay kits and Qubit 1X dsDNA HS Assay kits on a Qubit 4 Fluorometer, and analysed using Agilent DNA 1000 chips (Agilent Technologies, Santa Clara, California, USA) and Agilent HS DNA chips (Agilent) on an Agilent 2100 Bioanalyzer System.

Oh S.J., Pimentel M., Leite G.G., Celly S., Villanueva-Millan M.J., Lacsina I., Chuang B., Parodi G., Morales W., Weitsman S., Singer-Englar T., Barlow G.M., Zhai J., Pichestshote N., Rezaie A., Mathur R, & Pimentel M. (2020). Acute appendicitis is associated with appendiceal microbiome changes including elevated Campylobacter jejuni levels. BMJ Open Gastroenterology, 7(1), e000412.

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RNA-seq Analysis of PSC-CMs Differentiation

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Total RNA was extracted from the PSC-CMs at different time points of differentiation according to the manufacturer’s protocol for Direct-zol RNA extraction kit (Zymo Research), whereas RNA from mouse hearts was isolated by standard acid guanidinium thiocyanate-phenol-chloroform extraction using TRIzol. The quantity of the RNA samples was determined by Qubit 4 Fluorometer (Thermo Fisher Scientific). cDNA was prepared by QuantSeq. 3′ mRNA-Seq library prep kit FW for Illumina (Lexogen) according to the manufacturer’s protocol using ~500 ng total RNA per samples. Library concentrations and size-distribution were then confirmed by Qubit 4 Fluorometer and Agilent 2100 Bioanalyzer (Agilent Technologies), respectively. cDNA library was pooled from each cDNA sample and sequenced by the Illumina NextSeq (75 cycles, single-end). Adapter and quality trimming was performed with BBDuk, then the trimmed reads were mapped to the GRCm38 mouse genome using STAR RNA-seq aligner^{49 (link)}. To count the mapped read, featureCounts was used^{50 (link)}. The read counts were normalized to transcript per million to show gene expression levels and/or compare expression levels between genes. To compare expression levels between samples, the read counts were normalized to regularized log (rlog) using DEseq. 2^{51 (link)}. To perform GO analysis, enrichGO function in clusterProfiler was used^{52 (link)}.

Chanthra N., Abe T., Miyamoto M., Sekiguchi K., Kwon C., Hanazono Y, & Uosaki H. (2020). A Novel Fluorescent Reporter System Identifies Laminin-511/521 as Potent Regulators of Cardiomyocyte Maturation. Scientific Reports, 10, 4249.

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16S Library Preparation for Small Bowel and Stool

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16S library preparation for DNAs from small bowel aspirates and stool was performed according to the Illumina (Illumina, San Diego, CA, USA) protocol https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf as described previously [15 (link)], using the gene-specific primers S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21 published and validated by Klindworth et al. [16 (link)] to amplify the V3 and V4 regions. The final libraries were quantified using Qubit 1X dsDNA HS Assay kits on a Qubit 4 Fluorometer and analyzed using Agilent DNA 1000 chips (Agilent Technologies, Santa Clara, CA) on an Agilent 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA).

Leite G.G., Weitsman S., Parodi G., Celly S., Sedighi R., Sanchez M., Morales W., Villanueva-Millan M.J., Barlow G.M., Mathur R., Lo S.K., Jamil L.H., Paski S., Rezaie A, & Pimentel M. (2020). Mapping the Segmental Microbiomes in the Human Small Bowel in Comparison with Stool: A REIMAGINE Study. Digestive Diseases and Sciences, 65(9), 2595-2604.

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16S rRNA Metagenomics Sequencing

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DNA from nasopharyngeal swab samples was isolated using the QIAamp DNA Mini Kit (QIAgen) following the protocol recommended by the manufacturer. Sequencing libraries were prepared according to the 16S Metagenomic Sequencing Library Preparation protocol distributed by Illumina. Briefly, the sequence spanning the hypervariable regions V3 and V4 of the 16S rRNA gene was amplified through PCR. Amplicons were quantified using a Qubit 4 Fluorometer (Qubit dsDNA HS Assay Kit) validated by 4200 TapeStation (Agilent company). Amplicons were sequenced with Illumina MiSeq System using the 2 × 300 bp cartridge. The quality of raw sequences was assessed by FastQC software.

Ventero M.P., Cuadrat R.R., Vidal I., Andrade B.G., Molina-Pardines C., Haro-Moreno J.M., Coutinho F.H., Merino E., Regitano L.C., Silveira C.B., Afli H., López-Pérez M, & Rodríguez J.C. (2021). Nasopharyngeal Microbial Communities of Patients Infected With SARS-CoV-2 That Developed COVID-19. Frontiers in Microbiology, 12, 637430.

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Quantifying RNAi-Mediated Gene Knockdown

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RNAi lines were crossed with the tubGal80ts; tubGal4/Tm6B line, and shifted to the permissive temperature (29 °C) at day 1 post eclosion. Flies were collected after 7 days. For each group (control or KD flies) 6x adult flies were collected and RNA prepared using RNeasy Mini kit (Qiagen). RNA quantification was determined using the Qubit 4 Fluorometer and RNA quality assessed on an Agilent 2200 TapeStation (all RIN values were above 9.8). cDNA synthesis was carried out using SensiFAST cDNA Synthesis kit (Bioline). Droplet Digital PCR (ddPCR was performed using predesigned Taqman gene expression assays for Rnp-4f (Dm01799055_g1, Thermo Fisher Scientific) or Rpl32 (Dm02151827_g1, Thermo Fisher Scientific). Expression of Rnp4f was calculated as a percentage of the control in each RNAi line KD.

Ayers K.L., Eggers S., Rollo B.N., Smith K.R., Davidson N.M., Siddall N.A., Zhao L., Bowles J., Weiss K., Zanni G., Burglen L., Ben-Shachar S., Rosensaft J., Raas-Rothschild A., Jørgensen A., Schittenhelm R.B., Huang C., Robevska G., van den Bergen J., Casagranda F., Cyza J., Pachernegg S., Wright D.K., Bahlo M., Oshlack A., O’Brien T.J., Kwan P., Koopman P., Hime G.R., Girard N., Hoffmann C., Shilon Y., Zung A., Bertini E., Milh M., Ben Rhouma B., Belguith N., Bashamboo A., McElreavey K., Banne E., Weintrob N., BenZeev B, & Sinclair A.H. (2023). Variants in SART3 cause a spliceosomopathy characterised by failure of testis development and neuronal defects. Nature Communications, 14, 3403.

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Sonicated DNA for Illumina Sequencing

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After SPRI bead purification of the crosslink-reversed samples, we transferred DNA from each to Covaris microTUBE AFA Fiber Snap-Cap tubes (Covaris catalog no. 520045) and sonicated to an average length of 400 ± 85 base pairs using a Covaris ME220 Focused-Ultrasonicator. Temperature was held stably at 6 °C and treatment lasted 65 s per sample with a peak power of 50 W, 10% duty factor and 200 cycles per burst. The average fragment length and distribution of sheared DNA was determined by capillary electrophoresis using an Agilent FragmentAnalyzer 5200 and HS NGS Fragment Kit (Agilent catalog no. DNF-474–0500). We ran sheared DNA samples twice through the NEBNext Ultra II DNA Library Prep Kit for Illumina (catalog no. E7645S) End Preparation and Adapter Ligation steps with custom Y adapters to produce library preparation replicates. We purified ligation products via SPRI beads before Biotin enrichment using Dynabeads MyOne Streptavidin C1 beads (ThermoFisher catalog no. 65002).
We performed indexing PCR on streptavidin beads using KAPA HiFi HotStart ReadyMix (catalog no. KK2602) and PCR products were isolated by SPRI bead purification. We quantified the libraries by Qubit 4 fluorometer and FragmentAnalyzer 5200 HS NGS Fragment Kit (Agilent catalog no. DNF-474-0500) before pooling for sequencing on an Illumina HiSeq X at Fulgent Genetics.

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RNA Isolation and Quality Assessment

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The Macherey Nagel NucleoSpin totalRNA FFPE XS kit was used for RNA isolation (see Supplementary Methods). After RNA isolation, RNA quantity was measured using the Qubit RNA HS (High Sensitivity) Assay Kit with a Qubit 4 Fluorometer and RNA quality using the Agilent High Sensitivity RNA ScreenTape with an Agilent 4150 TapeStation. The DV₂₀₀ value of the sample (i.e., the percentage of fragments ≥ 200 bases in length) was calculated as recommended by Illumina. Samples with DV₂₀₀ values > 15% were considered as viable samples for library preparation.

Paul E.D., Huraiová B., Valková N., Birknerova N., Gábrišová D., Gubova S., Ignačáková H., Ondris T., Bendíková S., Bíla J., Buranovská K., Drobná D., Krchnakova Z., Kryvokhyzha M., Lovíšek D., Mamoilyk V., Mančíková V., Vojtaššáková N., Ristová M., Comino-Méndez I., Andrašina I., Morozov P., Tuschl T., Pareja F, & Čekan P. (2023). Multiplexed RNA-FISH-guided Laser Capture Microdissection RNA Sequencing Improves Breast Cancer Molecular Subtyping, Prognostic Classification, and Predicts Response to Antibody Drug Conjugates. medRxiv.

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