Qubit 3.0 fluorometer

Manufactured by Qiagen

The Qubit 3.0 Fluorometer is a compact and easy-to-use instrument designed for accurate and sensitive quantification of nucleic acids and proteins. It utilizes fluorescence-based detection methods to provide rapid and reliable measurements of sample concentrations. The Qubit 3.0 Fluorometer is a core laboratory tool for researchers and scientists in various fields.

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Lab products found in correlation

Ncounter neuropathology panel, by NanoString (2 mentions) Ncounter sprint cartridge, by NanoString (2 mentions) Micro bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Rnase free water, by Qiagen (2 mentions) Qubit dsdna broad range kit, by Qiagen (2 mentions) Nucleospin gel and pcr clean up column, by Macherey-Nagel (2 mentions) Qubit rna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions) Superscript 3 reverse transcription system, by Thermo Fisher Scientific (1 mentions) Kapa library quantification kit, by Agilent Technologies (1 mentions) Qiaseq stranded rna library kits, by Qiagen (1 mentions) Ribo off rrna depletion kit human mouse rat, by Vazyme (1 mentions) Agencourt rnaclean xp, by Beckman Coulter (1 mentions) Qubit 3.0 fluorometer, by Agilent Technologies (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Nextseq 500 platform, by Illumina (1 mentions) Qiaamp dna mini kit, by Qiagen (1 mentions)

6 protocols using qubit 3.0 fluorometer

NanoString nCounter Gene Expression Analysis

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Gene expression analysis was performed using the NanoString nCounter^® Neuropathology panel (#XT-CSO-MNROP1-12, NanoString Technologies). Two panels were used: one loaded with RNA isolated from BDEVs (that were filtered during the preparation of EVs); and another one where all the samples bypassed the RNA isolation (“non-isolated”, NI) but some were filtered (F) during BDEVs preparation, while others were not (NF).
To run the first panel, 50 ng of previously isolated RNA measured by Qubit™ RNA High Sensitivity Assay Kit (Thermo Fisher) using the 3.0 QuBit Fluorometer, were mixed with RNAse–free water (Qiagen) up to 5 µL. Samples were hybridized for 18 h and mixed with 15 µL of RNAse-free water to be loaded on the nCounter^® Sprint Cartridge (#LBL-10038-01, NanoStringTechnologies), following the instructions of the company. The analysis was run for 6 h.
For direct loading of EV lysates (i.e., no previous RNA isolation, NI), frozen EVs were resuspended in RLT lysis buffer and RNAse-free water in a ratio of 1:3 and loaded based on protein concentration measured by Micro BCA Protein Assay Kit (Thermo Scientific). 2.8 μg of proteins were loaded.

Bub A., Brenna S., Alawi M., Kügler P., Gui Y., Kretz O., Altmeppen H., Magnus T, & Puig B. (2022). Multiplexed mRNA analysis of brain-derived extracellular vesicles upon experimental stroke in mice reveals increased mRNA content with potential relevance to inflammation and recovery processes. Cellular and Molecular Life Sciences, 79(6), 329.

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Gene Expression Analysis of BDEVs

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Gene expression analysis was performed by using the NanoString nCounter® Neuropathology panel (#XT-CSO-MNROP1-12, NanoString Technologies). Two panels were used: one loaded with RNA isolated from BDEVs (that were filtered during the preparation of EVs); and another one where all the samples bypassed the RNA isolation ("non-isolated", NI) but some were filtered during the BDEVs preparation (F) and the others were not filtered (NF) during preparation. To run the first panel, 50 ng of previously isolated RNA measured by Qubit TM RNA High Sensitivity Assay Kit (Thermo Fisher) using the 3.0 QuBit Fluorometer, were mixed with RNAse-free water (Qiagen) up to 5 µL. Samples were hybridized for 18 h and mixed with 15 µL of RNAse-free water to be loaded on the nCounter® Sprint Cartridge (#LBL-10038-01, NanoStringTechnologies), following the instructions of the company. The analysis was run for 6 h. For direct loading of EV lysates (i.e., no previous RNA isolation, NI), frozen EVs were resuspended in RLT lysis buffer and RNAse-free water in a ratio of 1:3 and loaded based on protein concentration measured by Micro BCA Protein Assay Kit (Thermo Scientific). 2,8 µg of proteins were loaded.

Bub A., Brenna S., Alawi M., Kügler P., Gui Y., Kretz O., Altmeppen H.C., Magnus T., & Puig B. (2021). Multiplexed mRNA analysis of brain-derived extracellular vesicles upon experimental stroke in mice reveals increased mRNA content related to inflammation and recovery processes.

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RNA Extraction and RNA-seq Library Preparation

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RNA was extracted from 1 × 10⁷ cells with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and the concentration was measured with a Qubit 3.0 Fluorometer. For NGS, 1 µg of RNA was treated with the Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Vazyme) according to the manufacturer’s protocol and the concentration was measured again. Subsequently, 10–100 ng of rRNA depleted RNA was used to prepare RNA-seq library with QIAseq Stranded RNA Library Kits (Qiagen). Size distribution of DNA fragments of final libraries were confirmed using an Agilent Fragment analyzer with the DNF 474 kit and libraries were quantified with a Qubit 3.0 Fluorometer and the KAPA library quantification kit. All the DNA libraries were pooled and sequenced on the Illumina NextSeq 500 platform.
For RT-qPCR, extracted RNA was subjected to DNase I (NEB) treatment and purified with Agencourt® RNAClean™ XP (Beckman Coulter) before first-strand synthesis. First-strand synthesis was carried out by using Superscript III Reverse Transcription System (Thermo Scientific) according to the manufacturer’s protocol. cDNA was then analyzed by qPCR on LightCycler® 480 Instrument II. Primers used in this study are listed in Supplementary Data 4.

Zhou X., Sam T.W., Lee A.Y, & Leung D. (2021). Mouse strain-specific polymorphic provirus functions as cis-regulatory element leading to epigenomic and transcriptomic variations. Nature Communications, 12, 6462.

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Vaginal Microbiome Profiling using Nanopore Sequencing

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DNA was isolated from vaginal samples using QIAamp DNA Mini Kit (Qiagen), following the manufacturer’s protocol for Gram-positive and difficult-to-lyse bacteria, and quantified using Qubit 3.0 Fluorometer. 16S rRNA genes in each sample were amplified with the primers, B27F (TTTCTGTTGGTGCTGATATTGCAGRGTTYGATYMTGGCTCAG) and B1492R (ACTTGCCTGTCGCTCTATCTTCRGYTACCTTGTTACGACTT), which were modified from S-D-bact-0008-c-S20 and S-*-Univ-1492-a-A-19.16 (link) DNA libraries, each containing 12 different clinical samples, were prepared using PCR Barcoding Kit - EXP-PBC096 and Ligation Sequencing Kit 1D - SQK-LSK108, and sequenced on MinION MK I with SpotON Flow Cell Mk I FLO-MIN106 R9.4 (Oxford Nanopore Technologies). Sequencing reads were live base called locally using the MinKNOW protocol - NC_48Hr_Sequencing_Run_FLO-MIN106_ SQK-LSK108.py. Sequence data have been submitted to the ENA database with accession number PRJEB41336.

Kerry-Barnard S., Zhou L., Phillips L., Furegato M., Witney A.A., Sadiq S.T, & Oakeshott P. (2022). Vaginal microbiota in ethnically diverse young women who did or did not develop pelvic inflammatory disease: community-based prospective study. Sexually Transmitted Infections, 98(7), 503-509.

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Amplification and Sequencing of TiLV

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Partial regions of the TiLV segment 1 genome were amplified by semi‐nested RT‐PCR as described previously (Taengphu et al., 2020 (link)). Five microliters of the second round PCR products were analysed by electrophoresis on a 1% agarose gel stained with ethidium bromide solution. The remaining 20 µl reaction volume from the second round PCR was purified for each sample on a NucleoSpin Gel and PCR Clean‑up column (Macherey‐Nagel) and eluted with 20 μl of the kit elution buffer (5 mM Tris‐HCl, pH 8.5). The purified products were quantified using Qubit dsDNA Broad Range kit (Qiagen) with a Qubit 3.0 fluorometer prior to Nanopore multiplex library preparation.

Delamare‐Deboutteville J., Taengphu S., Gan H.M., Kayansamruaj P., Debnath P.P., Barnes A., Wilkinson S., Kawasaki M., Vishnumurthy Mohan C., Senapin S, & Dong H.T. (2021). Rapid genotyping of tilapia lake virus (TiLV) using Nanopore sequencing. Journal of Fish Diseases, 44(10), 1491-1502.

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Partial TiLV Genome Amplification

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Partial regions of the TiLV segment 1 genome were amplified by semi-nested RT-PCR as described previously (Taengphu et al., 2020) (link). Five microliters of the second round PCR products were analyzed by electrophoresis on a 1% agarose gel stained with ethidium bromide solution. The remaining 20 µL reaction volume from the second round PCR was purified for each sample on a NucleoSpin Gel and PCR Clean up column (Macherey-Nagel) and eluted with 20 μL of the kit elution buffer (5 mM Tris-HCl, pH 8.5). The purified products were quantified using Qubit dsDNA Broad Range kit (Qiagen) with a Qubit 3.0 fluorometer prior to Nanopore multiplex library preparation.

Delamare‐Deboutteville J., Taengphu S., Gan H.M., Kayansamruaj P., Debnath P.P., Barnes A.C., Wilkinson S., Kawasaki M., Mohan C.V., Senapin S., & Dong H.T. (2021). Rapid genotyping of tilapia lake virus (TiLV) using Nanopore sequencing.

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