Qubit 3.0 instrument

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The Qubit 3.0 instrument is a fluorometer designed for accurate quantification of DNA, RNA, and protein samples. It utilizes fluorescent dye-based assays to provide precise measurements of sample concentrations.

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Lab products found in correlation

Qubit dsdna high sensitivity assay, by Thermo Fisher Scientific (4 mentions) Random hexamer, by Thermo Fisher Scientific (3 mentions) Ampure xp bead, by Beckman Coulter (2 mentions) Miseq platform, by Illumina (2 mentions) Gel and pcr purification kit, by Macherey-Nagel (2 mentions) Ligation sequencing kit, by Oxford Nanopore (2 mentions) Native barcoding kit, by Oxford Nanopore (2 mentions) Qubit protein assay kit, by Thermo Fisher Scientific (2 mentions) Zetaview, by Particle Metrix (2 mentions) Exoeasy maxi kit, by Qiagen (2 mentions) Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions) Protoscript 2 first strand cdna synthesis kit, by New England Biolabs (1 mentions) Dna prep kit, by Illumina (1 mentions) Qubit double strand dna dsdna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions) Nextera xt dna library prep kit, by Illumina (1 mentions) Qubit dsdna broad range assay kit, by Thermo Fisher Scientific (1 mentions) Protoscript 2 reverse transcriptase, by New England Biolabs (1 mentions) Q5 high fidelity dna polymerase kit, by New England Biolabs (1 mentions) Ampure bead, by Beckman Coulter (1 mentions) Nextera xt library prep kit, by Illumina (1 mentions)

Close spelling variants of this product

Qubit 3.0 (396 citations) Qubit instrument (95 citations) QUBIT 3.0 fluorometer instrument (4 citations)

13 protocols using qubit 3.0 instrument

SARS-CoV-2 Whole Genome Amplification Protocol

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Viral RNA was used for cDNA transcription using Protoscript II First Strand cDNA synthesis Kit (New England Biolabs, UK) and random hexamers (Thermo Fisher Scientific, USA). Whole genome amplification was performed by multiplex PCR using SARS-CoV-2 primers described previously (https://artic.network/ncov-2019) and Q5 High-Fidelity DNA polymerase (New England Biolabs, UK) [31 (link)]. PCR conditions have been previously reported (https://artic.network/ncov-2019). PCR products were purified using the 1x AMPure XP beads (Beckman Coulter, United Kingdom) and quantified using fluorimeter with the Qubit dsDNA High Sensitivity assay on the Qubit 3.0 instrument (Life Technologies, USA).

Ferreira G.M., Claro I.M., Grosche V.R., Cândido D., José D.P., Rocha E.C., de Moura Coletti T., Manuli E.R., Gaburo N J.r., Faria N.R., Sabino E.C., de Jesus J.G, & Jardim A.C. (2022). Molecular characterization and sequecing analysis of SARS-CoV-2 genome in Minas Gerais, Brazil. Biologicals, 80, 43-52.

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NGS Amplicon Sequencing for Discordant Samples

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Samples with discordant serology/PCR results or with multiple or very tiny bands on the agarose gel, making them not feasible for Sanger sequencing, were submitted to NGS amplicon sequencing as follows.
Briefly, PCR reactions were purified using a Gel and PCR purification kit (Macherey-Nagel). PCR amplified DNA was quantified with a fluorimetric method, Qubit double-strand DNA (dsDNA) High Sensitivity assay kit on a Qubit 3.0 instrument (Life Technologies, Leominster, MA, USA) and subjected to tagmentation, amplification, and indexing, using the Illumina DNA prep Kit and IDT for Illumina UD indexes (Illumina, San Diego, CA, USA), according to the manufacturer’s protocol. Libraries were then normalized to a 4 nM concentration, pooled, and denatured with 0.2 N sodium acetate. The 12.5 pM paired-end library was spiked with 5% PhiX control and sequenced on an Illumina Miseq platform, using a V2-500 cycles chemistry.

Colitti B., Daif S., Choukri I., Scalas D., Jerre A., El Berbri I., Fassi Fihri O, & Rosati S. (2024). Serological and Molecular Characterization of Small Ruminant Lentiviruses in Morocco. Animals : an Open Access Journal from MDPI, 14(4), 550.

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Whole Genome Sequencing of K. aerogenes CRKA317

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The K. aerogenes CRKA317 was selected for whole genome sequencing (WGS). The Nextera XT DNA Library Prep Kit (Illumina, San Diego, California, United States) was utilized to conduct the library preparation using 1 ng of DNA as our material to sequence. A limited cycle polymerase chain reaction (PCR) program was employed to amplify the libraries introducing Index 1 (i7) adapters, Index 2 (i5) adapters, and the requisite sequences for generating sequencing clusters. The amplified library was purified using 0.6 x Agencourt AMPure XP beads (Beckman Coulter, Brea, California, USA). The quality of the library and the size of fragmented DNA was evaluated on a 1.5% electrophoresis agarose gel and quantified using a fluorometric method involving the Qubit® 3.0 instrument and the Qubit® dsDNA Broad Range Assay Kit (Life Technologies, Carlsbad, California, United States). The resulting library concentrations were subsequently normalized to 4 nM using a standard dilution method. The libraries were then combined, denatured with 0.2 N sodium hydroxide (NaOH), and diluted to attain a final concentration of 1.8 pM. To ensure the run’s accuracy and control, a PhiX control was added to achieve a final concentration of 1.5 pM. The sequencing run involved a paired-end run comprising 75 cycles for each read (2 × 75), plus up to eight cycles for two index reads.

Rodrigues S.H., Nunes G.D., Soares G.G., Ferreira R.L., Damas M.S., Laprega P.M., Shilling R.E., Campos L.C., da Costa A.S., Malavazi I., da Cunha A.F, & Pranchevicius M.C. (2024). First report of coexistence of blaKPC-2 and blaNDM-1 in carbapenem-resistant clinical isolates of Klebsiella aerogenes in Brazil. Frontiers in Microbiology, 15, 1352851.

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Multiplex PCR Amplification of DENV-1 and ZIKV

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The extracted RNAs that were positive for DENV-1 and ZIKV were submitted to whole-genome amplification using a tiling, multiplex PCR approach that has been previously developed [36 (link)]. Briefly, the sample was converted to cDNA using random hexamers (Invitrogen; Carlsbad CA, USA) and ProtoScript II Reverse Transcriptase (New England BioLabs; Ipswich, MA, USA) according to the manufacturer's instructions. The cDNA was then amplified with a multiplex PCR assay designed from Primal Scheme using as input the “ZikaAsian” scheme for ZIKV (https://github.com/zibraproject) and the one described by Quick et al. (2017) for DENV-1 [36 (link)]. An 80% consensus generated from a reference alignment of DENV-1 sequences was used as input to Primal Scheme. PCR was performed using the Q5 High-Fidelity DNA polymerase (NEB). PCR products were cleaned-up using a 1:1 ratio of AMPure XP beads (Beckman Coulter, Brea, CA) and quantified using fluorimetry with the Qubit dsDNA High Sensitivity Assay on the Qubit 3.0 instrument (Life Technologies).

Ricas Rezende H., Malta Romano C., Morales Claro I., Santos Caleiro G., Cerdeira Sabino E., Felix A.C., Bissoli J., Hill S., Rodrigues Faria N., Cardoso da Silva T.C., Brioschi Santos A.P., Cerutti Junior C, & Vicente C.R. (2020). First report of Aedes albopictus infected by Dengue and Zika virus in a rural outbreak in Brazil. PLoS ONE, 15(3), e0229847.

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SARS-CoV-2 Genome Sequencing from Hospital Samples

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Biological samples from professionals with positive RT-PCR results collected in different sectors of the hospital were sequenced to assess clusters and the lineage of SARS-CoV-2. Contact tracing was not performed in this sample and RT-PCR for patients was not available for the research.
After extraction of viral RNA, cDNA synthesis was performed using the Protoscript II First Strand transcription kit (New England Biolabs) and random hexamers (Thermo Fisher Scientific). Amplification of the total genome was carried o\ut using the multiplex PCR reaction with the primers designed for the amplification of the complete genome of SARS-CoV-2 (https://artic.network/ncov-2019), together with the Q5 High-Fidelity DNA Polymerase Kit (New England Biolabs). The PCR conditions have been described previously (https://artic.network/ncov-2019). Amplicons formed after the PCR reaction were purified using magnetic beads (1 × AMPure XP, Beckman Coulter). After purification, the product was quantified using fluorometry techniques with Qubit dsDNA high sensitivity reagents, and reading was performed using the Qubit 3.0 instrument (Life Technologies).

Teixeira Mendes E., Neto D.G., Ferreira G.M., Valença I.N., Lima M.P., de Freitas M.F., Donalisio M.R., Melo M.C., Lazari C., Goes J., Morales I., Jardim A.C., Andrade dos Santos P., Franco L.A., Sabino E.C, & Costa S.F. (2022). Impact of COVID-19 RT-PCR testing of asymptomatic health care workers on absenteeism and hospital transmission during the pandemic. American Journal of Infection Control, 51(3), 248-254.

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Chikungunya Virus Nanopore Sequencing

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Extracted RNA from positive CHIKV samples were provided by collaborating LACENs and submitted to cDNA synthesis and PCR, using a sequencing protocol (primers sequences in Supplementary Data 1) based on multiplex PCR-tiling amplicon approach design for MinION nanopore sequencing^{74 (link)}. All reactions were performed at biosafety level 2 facilities and using no template controls. PCR products were purified using 1x AMpure beads Beckman Coulter, UK) and quantified using Qubit 3.0 instrument (Life Technologies) and the Qubit dsDNA High Sensitivity assay. DNA library preparation was performed on all amplified samples using the Ligation Sequencing Kit (Oxford Nanopore Technologies). Individual samples were barcoded using the Native Barcoding Kit (NBD104, Oxford Nanopore Technologies, Oxford, UK). Sequencing library was loaded onto a R9.4 flow cell and data were collected for up to 48 sequencing hours.

Xavier J., Alcantara L.C., Fonseca V., Lima M., Castro E., Fritsch H., Oliveira C., Guimarães N., Adelino T., Evaristo M., Rodrigues E.S., Santos E.V., de La-Roque D., de Moraes L., Tosta S., Neto A., Rosewell A., Mendonça A.F., Leite A., Vasconcelos A., Silva de Mello A.L., Vasconcelos B., Montalbano C.A., Zanluca C., Freitas C., de Albuquerque C.F., Duarte dos Santos C.N., Santos C.S., dos Santos C.A., Gonçalves C.C., Teixeira D., Neto D.F., Cabral D., de Oliveira E.C., Noia Maciel E.L., Pereira F.M., Iani F., de Carvalho F.P., Andrade G., Bezerra G., de Castro Lichs G.G., Pereira G.C., Barroso H., Franz H.C., Ferreira H., Gomes I., Riediger I.N., Rodrigues I., de Siqueira I.C., Silva J., Rico J.M., Lima J., Abrantes J., do Nascimento J.P., Wasserheit J.N., Pastor J., de Magalhães J.J., Luz K.G., Lima Neto L.G., Frutuoso L.C., da Silva L.B., Sena L., de Sousa L.A., Pereira L.A., Demarchi L., Câmara M.C., Astete M.G., Almiron M., Lima M., Umaki Zardin M.C., Presibella M.M., Falcão M.B., Gale M J.r., Freire N., Marques N., de Moura N.F., Almeida Da Silva P.E., Rabinowitz P., da Cunha R.V., Trinta K.S., do Carmo Said R.F., Kato R., Stabeli R., de Jesus R., Hans Santos R., Kashima S., Slavov S.N., Andrade T., Rocha T., Carneiro T., Nardy V., da Silva V., Carvalho W.G., Van Voorhis W.C., Araujo W.N., de Filippis A.M, & Giovanetti M. (2023). Increased interregional virus exchange and nucleotide diversity outline the expansion of chikungunya virus in Brazil. Nature Communications, 14, 4413.

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Nanopore Sequencing of CHIKV RNA

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Extracted RNA from positive CHIKV samples were provided by collaborating LACENs and submitted to cDNA synthesis and PCR, using a sequencing protocol based on multiplex PCR tiling amplicon approach design for MinION nanopore sequencing^{62 (link)}. All reactions were performed at biosafety level 2 facilities and using no template controls. PCR products were purified using 1x AMpure beads Beckman Coulter, UK) and quantified using Qubit 3.0 instrument (Life Technologies) and the Qubit dsDNA High Sensitivity assay. DNA library preparation was performed on all amplified samples using the Ligation Sequencing Kit (Oxford Nanopore Technologies). Individual samples were barcoded using the Native Barcoding Kit (NBD104, Oxford Nanopore Technologies, Oxford, UK). Sequencing library was loaded onto a R9.4 flow cell and data were collected for up to 48 sequencing hours.

Xavier J., Alcantara L., Fonseca V., Lima M., Castro E., Fritsch H., Oliveira C., Guimarães N., Adelino T., Evaristo M., Rodrigues E.S., Santos E.V., de La-Roque D., de Moraes L., Tosta S., Neto A., Rosewell A., Mendonça A.F., Leite A., Vasconcelos A., Silva de Mello A.L., Vasconcelos B., Montalbano C.A., Zanluca C., Freitas C., de Albuquerque C.F., Duarte dos Santos C.N., Santos C.S., dos Santos C.A., Maymone Gonçalves C.C., Teixeira D., Neto D.F., Cabral D., de Oliveira E.C., Noia Maciel E.L., Pereira F.M., Iani F., de Carvalho F.P., Andrade G., Bezerra G., de Castro Lichs G.G., Pereira G.C., Barroso H., Ferreira Franz H.C., Ferreira H., Gomes I., Riediger I.N., Rodrigues I., de Siqueira I.C., Silva J., Rico J.M., Lima J., Abrantes J., do Nascimento J.P., Wasserheit J.N., Pastor J., de Magalhães J.J., Luz K.G., Lima Neto L.G., Frutuoso L.C., da Silva L.B., Sena L., de Sousa L.A., Pereira L.A., Demarchi L., Câmara M.C., Astete M.G., Almiron M., Lima M., Umaki Zardin M.C., Presibella M.M., Falcão M.B., Gale M J.r., Freire N., Marques N., de Moura N.F., Almeida Da Silva P.E., Rabinowitz P., da Cunha R.V., Trinta K.S., do Carmo Said R.F., Kato R., Stabeli R., de Jesus R., Santos R.H., Haddad S.K., Slavov S.N., Andrade T., Rocha T., Carneiro T., Nardy V., da Silva V., Carvalho W.G., Van Voorhis W.C., Araujo W.N., de Filippis A.M, & Giovanetti M. (2023). Increased interregional virus exchange and nucleotide diversity outline the expansion of the chikungunya virus ECSA lineage in Brazil. medRxiv.

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FFPE Tissue DNA and RNA Extraction

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Formalin‐fixed paraffin embedded (FFPE) tumor blocks were utilized for molecular analysis following quality review of a hematoxylin and eosin (H&E) slide. A reference pathologist (H.S.) identified specific tumor‐rich areas which were then macrodissected from 10‐µm tissue sections using a sterile razor blade followed by deparaffinization in 1 ml of xylene. Genomic DNA and total RNA were isolated using the Qiagen AllPrep FFPE Tissue Kit (QIAGEN) followed by fluorometric quantification using a Qubit 3.0 instrument (Life Technologies). RNA integrity was assessed using the Agilent TapeStation on a High Sensitivity RNA ScreenTape.

Weberpals J.I., Pugh T.J., Marco‐Casanova P., Goss G.D., Andrews Wright N., Rath P., Torchia J., Fortuna A., Jones G.N., Roudier M.P., Bernard L., Lo B., Torti D., Leon A., Marsh K., Hodgson D., Duciaume M., Howat W.J., Lukashchuk N., Lazic S.E., Whelan D, & Sekhon H.S. (2021). Tumor genomic, transcriptomic, and immune profiling characterizes differential response to first‐line platinum chemotherapy in high grade serous ovarian cancer. Cancer Medicine, 10(9), 3045-3058.

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Exosome Isolation and Characterization

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Collection of EVs was carried out by seeding 1 × 10⁶ cells per 10 cm² dish and incubating overnight. Following the overnight incubation, cells were washed in phosphate-buffered saline (PBS) and fresh media containing exosome-free fetal bovine serum (#A2720801, Gibco) applied. Cells were incubated for 48 h under standard conditions. After 48 h, the media was collected and vesicles isolated using the exoEasy Maxi kit (#76064, Qiagen, Manchester, UK) and gravity flow chromatography (#qEV, Izon Science, Oxford, UK) following the manufacturers’ protocols to isolate vesicles of less than 200 nm and within the range considered to represent exosomes. EVs were characterised by electrophoresis and Brownian motion analysis using laser scattering microscopy (ZetaView, ParticleMetrix, Meerbusch, Germany). Total protein was determined using the Qubit Protein Assay Kit (#Q33211, ThermoFisher, Loughborough, UK) and the Qubit 3.0 instrument (ThermoFisher). EV protein markers were assessed by immunoblot as subsequently described in the Protein extraction and immunoblots section.

Probert C., Dottorini T., Speakman A., Hunt S., Nafee T., Fazeli A., Wood S., Brown J.E, & James V. (2018). Communication of prostate cancer cells with bone cells via extracellular vesicle RNA; a potential mechanism of metastasis. Oncogene, 38(10), 1751-1763.

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Isolation and Characterization of Extracellular Vesicles

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Collection of EVs was carried out by seeding 1x10⁶ cells per 10cm² dish and incubating overnight. Following the overnight incubation, cells were washed in PBS and fresh media containing exosome free fetal bovine serum (#A2720801, Gibco) applied. Cells were incubated for 48 hours under standard conditions. After 48 hours, the media was collected and vesicles isolated using the exoEasy Maxi kit (#76064, Qiagen, Manchester, UK) and gravity flow chromatography (#qEV, Izon Science, Oxford, UK) following the manufacturers’ protocols to isolate vesicles of less than 200nm and within the range considered to represent exosomes. EVs were characterised by electrophoresis and Brownian motion analysis using laser scattering microscopy (ZetaView, ParticleMetrix, Meerbusch, Germany). Total protein was determined using the Qubit Protein Assay Kit (#Q33211, ThermoFisher, Loughborough, UK) and the Qubit 3.0 instrument (ThermoFisher). EV protein markers were assessed by immunoblot as subsequently described in the Protein extraction and immunoblots section.

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