Qubit 2.0 fluorometer

Manufactured by Agilent Technologies

Sourced in United States

The Qubit 2.0 Fluorometer is a compact and accurate instrument for measuring the concentration of DNA, RNA, and protein samples. It utilizes fluorescent dyes that specifically bind to the target molecules, providing a sensitive and reliable method for quantification.

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143 protocols using qubit 2.0 fluorometer

Litchi Pulp Transcriptome Sequencing

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According to the changes in the total soluble sugar content in ‘Feizixiao’ litchi pulp in 2020, RNA was extracted from the pulp at 35, 63, and 69 DAA for transcriptome sequencing, with 3 biological replicates in each time period. A plant total RNA extraction kit (RNAprep Pure Plant Plus Kit, catalog No: DP441) from Tiangen Biochemical Technology Co., Ltd. was used for RNA extraction. The procedure was carried out according to the instructions provided in the kit. RNA integrity and DNA contamination were analyzed by agarose gel electrophoresis. RNA purity, concentration, and integrity were accurately measured on a NanoPhotometer spectrophotometer, a Qubit 2.0 Fluorometer, and an Agilent 2100 Bioanalyzer, respectively. Then, a cDNA library was constructed from the high-quality RNA by Wuhan Metwell Biotechnology Co. Ltd. After the library was constructed, real-time PCR, a Qubit 2.0 Fluorometer, and an Agilent 2100 Bioanalyzer were used for quality inspection. Transcriptome sequencing was performed on the Illumina HiSeq platform after the library was qualified. Then, the raw reads were filtered to remove low-quality reads with adapters and to obtain clean reads.

Peng J., Du J., Wuqiang M., Chen T., Shui X., Liao H., Lin X, & Zhou K. (2022). Transcriptomics‐based analysis of the causes of sugar receding in Feizixiao litchi (Litchi chinensis Sonn.) pulp. Frontiers in Plant Science, 13, 1083753.

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RNA Extraction and Sequencing Protocol

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RNA extraction was performed using the RNeasy Plus Mini kit (Qiagen) following the manufacturer’s protocol from sample prepared from the tip of the swab (see Figure 1). RNA purity was assessed using Qubit 2.0 fluorometer and Agilent TapeStation 2200 for RNA quantity and quality. Host RNA sequencing libraries were generated using Illumina TruSeq RNA Access, which converts total RNA into template molecules, followed by sequence-specific capture of coding RNA (25 ). The cDNA libraries were quantified using Qubit 2.0 fluorometer and quality examined using Agilent TapeStation 2200.The cDNA libraries were pooled at a final concentration 2.0pM. Cluster generation and paired-read, dual-indexed 75bp sequencing was performed on Illumina NextSeq 500.

Lopez S.M., Martin J.M., Johnson M., Kurs-Lasky M., Horne W.T., Marshall C.W., Cooper V.S., Williams J.V, & Shaikh N. (2019). A method of processing nasopharyngeal swabs to enable multiple testing. Pediatric research, 86(5), 651-654.

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Extraction and Quantification of cfDNA and gDNA

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cfDNA was extracted from aliquots (500 µl) of serum using the QIAamp circulating nucleic acid kit (Qiagen, Hilden, Germany) with the QIAvac 24 Plus vacuum manifold, following the manufacturer’s instructions. cfDNA purity was checked using an Agilent High Sensitivity DNA Kit and the Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, CA). When required, additional purification was performed using Agencourt AMPure XP (BeckMan Coulter, Brea, CA) to remove larger contaminating nucleic acid. cfDNA concentration was quantified with a Qubit 2.0 Fluorometer using the Agilent High Sensitivity DNA Kit (Agilent Technologies).
Tissue genomic DNA (gDNA) was extracted from formalin-fixed, paraffin-embedded (FFPE) tissues with the QIAamp DNA FFPE Tissue kit (Qiagen) according to the manufacturer’s instructions and eluted in a 50 μL volume. Purity of the extracted genomic DNA was assessed by electrophoresis of the DNA through a 1% agarose gel, and DNA concentration was quantified with a Qubit 2.0 Fluorometer using the Agilent High Sensitivity DNA Kit (Agilent Technologies).

Sung J.S., Chong H.Y., Kwon N.J., Kim H.M., Lee J.W., Kim B., Lee S.B., Park C.W., Choi J.Y., Chang W.J., Choi Y.J., Lee S.Y., Kang E.J., Park K.H, & Kim Y.H. (2017). Detection of somatic variants and EGFR mutations in cell-free DNA from non-small cell lung cancer patients by ultra-deep sequencing using the ion ampliseq cancer hotspot panel and droplet digital polymerase chain reaction. Oncotarget, 8(63), 106901-106912.

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RNA Extraction and Sequencing Protocol

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Fecal DNA Extraction and Sequencing

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DNA was extracted from 0.18–0.22 g of feces content of each sample with the DNeasy® PowerLyzer® PowerSoil® isolation kit (Qiagen) according to the manufacturer’s recommendations. The concentration of the extracted DNA was determined by fluorometric Quantification (Qubit 2.0 Fluorometer, Thermo Fisher Scientific) and stored at − 20 °C until use. Library preparation was preformed using the Nextera XT DNA Library Preparation Kit (Illumina, Int., San Diego, CA, USA) according to the manufacturer’s recommendation, by the DTU in-house facility (DTU Multi-Assay Core (DMAC), Technical University of Denmark). Size confirmation of the target was performed on a Bioanalyzer DNA 1000 chip (Agilent Technology, CA), and the DNA concentration was determined with Qubit 2.0 Fluorometer. DNA libraries were mixed in equimolar ratios. Sequencing was performed as a 150-bp paired-end run on HiSeq 3000/4000 (Illumina Int., San Diego, CA USA) at Novogene Europe’s facility following the manufacturer’s recommendations.

Wei S., Jespersen M.L., Baunwall S.M., Myers P.N., Smith E.M., Dahlerup J.F., Rasmussen S., Nielsen H.B., Licht T.R., Bahl M.I, & Hvas C.L. (2022). Cross-generational bacterial strain transfer to an infant after fecal microbiota transplantation to a pregnant patient: a case report. Microbiome, 10, 193.

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Gut Microbiome Analysis via 16S rRNA Sequencing

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DNA was extracted from collected fecal samples using the EZNA tool DNA extraction kit (Omega, D4015), and the V3-V4 variable region was amplified with 16s rRNA primers. According to Gene Tools Analysis Software mixed PCR products inequal ratios, purification of PCR products, and sequencing libraries were generated using NEBNEXT Ultra DNA. Library quality was assessed using the Qu bit 2.0 Fluorometer and the Agilent Bioanalyzer 2100 system, then the library was sequenced on an Illumina Hiseq platform, 280 bp paired-end reads were generated following the sequencing libraries to analyze intestinal gut microbiota composition.

Li L., Huang G., Chen T., Lin H., Xu R., Cheng J., Hu Y., Dai W, & Dong G. (2022). Fufang Fanshiliu Decoction Revealed the Antidiabetic Effect through Modulating Inflammatory Response and Gut Microbiota Composition. Evidence-based Complementary and Alternative Medicine : eCAM, 2022, 3255401.

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Laser Capture Microdissection and RNA Sequencing of Mouse Sebaceous Glands

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Mouse back skin from CR, GF, CR×CR F₁, and GF×GF F₁ was collected and fixed overnight in 4% paraformaldehyde (Fisher AAJ19943K2) at 4°C followed by paraffin embedding. Laser capture microdissection (LCM) was performed using the LMD 7000 system (Leica Microsystems). FFPE mouse skin was processed and cut onto a polyethylene naphthalate (PEN) slide designed for LCM processing (Leica 11505158). At least 1,000 SGs or 1,000,000 μm² of tissue was isolated to obtain enough material for RNA extraction. SG RNA was extracted from post-LCM tissue using a Qiagen All Prep DNA/RNA FFPE Kit (Qiagen 80234). RNA concentration was measured by Qubit fluorometric quantification (ThermoFisher Qubit 2.0 Fluorometer) and RNA quality measured via BioAnalyzer (Agilent 2100 Bioanalyzer Instrument). cDNA libraries were prepared using Illumina Stranded Total RNA Prep with Ribo-Zero Plus Kit (Illumina 20040529) with IDT for Illumina RNA UD Indexes, Set A (Illumina 20040553). Libraries were assessed for cDNA quantity and library quality using Qubit and BioAnalyzer. As necessary, an extra bead wash step was performed to remove excess primer dimers in the library and purify samples further. Samples were then pooled and sequenced on a Nextseq 550 using a NextSeq 500/550 High Output Kit v2.5 (150 Cycles) (Illumina 20024907).

Harris J.C., Trigg N.A., Goshu B., Yokoyama Y., Dohnalová L., White E.K., Harman A., Murga-Garrido S.M., Pan J.T., Bhanap P., Thaiss C.A., Grice E.A., Conine C.C, & Kambayashi T. (2024). The microbiota and T cells non-genetically modulate inherited phenotypes transgenerationally. Cell reports, 43(4), 114029.

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Ion RNA-Seq library construction

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Total 400 μl of each sample was used for nucleic acids extraction by using QIAamp® viral RNA mini Kit (Qiagen, Hilden, Germany) and the elution volume was 80 μl. Since both the DNA and RNA molecules are of interest to us, no DNase was used in our protocol. All nucleic acids were used for library construction. The OD260/280 value of total nucleic acids was measured by NanoDrop Spectrophotometer (ND-1000, Thermo Fisher, Wilmington, DE, USA). The RNA was specifically quantified by Qubit® 2.0 Fluorometer (Thermo Fisher, Wilmington, DE, USA). The libraries were constructed by using Ion Total RNA-Seq Kit v2 (Thermo Fisher, Wilmington, DE, USA). Total 30–50 ng of RNA was fragmented by RNase III (Thermo Fisher, Wilmington, DE, USA), then hybridized and ligated with the Ion Adaptor Mix v2 at 65 °C for 10 min, 30 °C for 30 min. The reverse transcribed single-stranded cDNA was amplified with Ion 5′ and 3′ PCR Primer v2. The sequencing libraries were qualified and quantified by using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit® 2.0 Fluorometer.

Xiao Y., Zhang L., Yang B., Li M., Ren L, & Wang J. (2019). Application of next generation sequencing technology on contamination monitoring in microbiology laboratory. Biosafety and Health, 1(1), 25-31.

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Directional RNA Sequencing Library Prep

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For long RNA library preparation a Ribo-zero Magnetic Gold rRNA removal kit (Epicenter, IIlumina Inc.) was used to remove ribosomal RNA from the total RNA. First strand synthesis was performed using NEBNext RNA first strand synthesis module (New England BioLabs Inc., Ipswich, MA, USA). Immediately thereafter, directional second strand synthesis was performed using NEBNExt Ultra Directional second strand synthesis kit. Following this, cDNAs were used for standard library preparation protocol using NEBNext DNA Library Prep Master Mix Set for Illumina. End-repair was performed followed by polyA addition and custom adapter ligation. After ligation, material was individually barcoded with unique in-house genomics service lab primers. Library quality and concentration were assessed by a Qubit 2.0 Fluorometer and DNA 1000 chips on an Agilent 2100 Bioanalyzer. Accurate quantification for sequencing applications was determined using the qPCR-based KAPA Biosystems Library Quantification kit (Kapa Biosystems, Inc., Woburn, MA). Each library was diluted to a final concentration of 12.5 nM and pooled equimolar prior to clustering. Paired-end sequencing was performed on all samples (100 bp paired-end directional reads). Raw reads were de-multiplexed using a bcl2fastq conversion software v1.8.3 (Illumina, Inc.) with default settings.

Jeppesen D.K., Fenix A.M., Franklin J.L., Higginbotham J.N., Zhang Q., Zimmerman L.J., Liebler D.C., Ping J., Liu Q., Evans R., Fissell W.H., Patton J.G., Rome L.H., Burnette D.T, & Coffey R.J. (2019). Reassessment of Exosome Composition. Cell, 177(2), 428-445.e18.

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RNA-Seq Library Construction and Sequencing

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RNA-Seq libraries were constructed from approximately 100 ng rRNA-depleted RNA using a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) or KAPA RNA HyperPrep Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Constructed sequencing libraries were quantified using a Qubit dsDNA HS Assay Kit with a Qubit 2.0 fluorometer and a TapeStation 2200 (Agilent, Santa Clara, CA, USA) equipped with a High Sensitivity D1000 Screen Tape (Agilent). The library was sequenced using an Illumina platform. Information on NGS (run recipe and instrument) is summarized in S6 Table.

Choe D., Szubin R., Poudel S., Sastry A., Song Y., Lee Y., Cho S., Palsson B, & Cho B.K. (2021). RiboRid: A low cost, advanced, and ultra-efficient method to remove ribosomal RNA for bacterial transcriptomics. PLoS Genetics, 17(9), e1009821.

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