Versant hiv 1 rna 3.0 assay

Manufactured by Siemens

Sourced in United Kingdom, United States, Germany

The Versant HIV-1 RNA 3.0 assay is a laboratory diagnostic tool used for the quantitative detection of human immunodeficiency virus type 1 (HIV-1) RNA in human plasma. The assay provides a measure of the viral load, which is a key indicator in the management of HIV-1 infection.

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Lab products found in correlation

Realtime hiv 1 assay, by Abbott (6 mentions) Facscalibur flow cytometer, by BD (6 mentions) Nuclisens hiv 1 rna qt assay, by Organon (5 mentions) Facscanto, by BD (3 mentions) Ficoll hypaque density gradient centrifugation, by Cytiva (3 mentions) Ficoll hypaque density gradient centrifugation, by GE Healthcare (2 mentions) Facscalibur, by BD (1 mentions) Trucount absolute count tubes, by BD (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Merck Group (1 mentions) Penicillin, by Merck Group (1 mentions) Streptomycin, by Merck Group (1 mentions) L glutamine, by Merck Group (1 mentions) Facscaliburtm system, by BD (1 mentions)

14 protocols using versant hiv 1 rna 3.0 assay

Quantification of HIV-1 RNA in Cells

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5 × 10⁶ cells from the Thy/Liv single cell suspension were stored as dry pellets at −80°C. Prior to bDNA assay cells were lysed mechanically in sterile disposable pestles and motor grinder (Kontes) in M guanidine HCl with 0.5% sodium N-lauroylsarcosine. RNA was then extracted with 500 uL 100% ethanol and pelleted at 12,000 × g for 20 min at 4°C. The RNA pellet was washed with 500 uL 70% ethanol and digested using supplied reagents according to manufacturer instructions of the VERSANT HIV-1 RNA 3.0 Assay (Siemens Healthcare Diagnostics). HIV-1 RNA is expressed as copies/10⁶ cells, and log₁₀ values were used to calculate geometric means.

Cham L.B., Torrez Dulgeroff L.B., Tal M.C., Adomati T., Li F., Bhat H., Huang A., Lang P.A., Moreno M.E., Rivera J.M., Galkina S.A., Kosikova G., Stoddart C.A., McCune J.M., Myers L.M., Weissman I.L., Lang K.S, & Hasenkrug K.J. (2020). Immunotherapeutic Blockade of CD47 Inhibitory Signaling Enhances Innate and Adaptive Immune Responses to Viral Infection. Cell reports, 31(2), 107494.

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Blood and PBMC Collection for HIV Immunology

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Blood samples were collected from study participants at enrollment, centrifuged to separate plasma, which was stored at −80°C until use. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (GE Healthcare, Little Chalfont/Buckinghamshire, UK) and cryopreserved for subsequent phenotypic and functional assays. PBMC cryopreservation and thawing were performed following protocols developed to obtain an optimal detection of antigen specific T-cell responses [23] (link)–[25] . For PHI subjects, samples were obtained at enrollment (baseline) and at 3, 6, 9, and 12 months post-infection. Samples used for immunophenotypic and functional analyses were obtained from subjects that remained HAART-naïve at the moment of sampling. In the case PHI subject needed to start HAART early, baseline samples were obtained before treatment instauration. Plasma VL (branched-DNA, Versant HIV-1 RNA 3.0 assay; Siemens Healthcare, Sudbury/Suffolk, UK) and CD4⁺ T-cell count (flow cytometry double platform, BD FACSCanto; BD Biosciences, San Diego/California, USA) were assessed in HIV-infected subjects.

Ghiglione Y., Falivene J., Ruiz M.J., Laufer N., Socías M.E., Cahn P., Giavedoni L., Sued O., Gherardi M.M., Salomón H, & Turk G. (2014). Early Skewed Distribution of Total and HIV-Specific CD8+ T-Cell Memory Phenotypes during Primary HIV Infection Is Related to Reduced Antiviral Activity and Faster Disease Progression. PLoS ONE, 9(8), e104235.

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Blood, Cells and Viral Load Quantification

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We collected 40 ml of whole blood. Upon centrifugation, plasma was recovered, and stored at −80C. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (Amersham, Sweden) and cryopreserved in liquid nitrogen for subsequent functional assays. Plasma viral load (VL) was determined by branched-DNA, Versant HIV-1 RNA 3.0 assay (Siemens Healthcare, UK) or by Abbott Real Time HIV-1 assay (Abbott Park, IL) depending on kit availability.
CD4+ and CD8+ T-cell counts were determined by flow cytometry (FACSCalibur; BD Biosciences, USA). Cellular immune activation was evaluated as the percentage of CD38- and/or HLA-DR-expressing CD4+ and CD8+ T cells by flow cytometry.

Salido J., Czernikier A., Trifone C., Polo M.L., Figueroa M.I., Urioste A., Cahn P., Sued O., Salomon H., Laufer N., Ghiglione Y, & Turk G. (2021). Pre-cART Immune Parameters in People Living With HIV Might Help Predict CD8+ T-Cell Characteristics, Inflammation Levels, and Reservoir Composition After Effective cART. Pathogens and Immunity, 6(2), 60-89.

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Quantification of HIV-1 RNA in Cells

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PBMC Isolation and Viral Load Measurement

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Forty ml of whole blood were collected from study participants, centrifuged to separate plasma, and stored at −80°C. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (Amersham, Sweden) and cryopreserved for subsequent functional assays. Plasma viral load (VL) was determined by branched-DNA, Versant HIV-1 RNA 3.0 assay (Siemens Healthcare, UK). CD4⁺ and CD8⁺ T-cell counts were determined using TruCount absolute-count tubes (BD Bioscences, USA) on a BD FACSCalibur flow cytometer.

Salido J., Ruiz M.J., Trifone C., Figueroa M.I., Caruso M.P., Gherardi M.M., Sued O., Salomón H., Laufer N., Ghiglione Y, & Turk G. (2018). Phenotype, Polyfunctionality, and Antiviral Activity of in vitro Stimulated CD8+ T-Cells From HIV+ Subjects Who Initiated cART at Different Time-Points After Acute Infection. Frontiers in Immunology, 9, 2443.

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ADCC Assay Using Plasma and PBMCs

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Plasma and peripheral blood mononuclear cells (PBMCs) were obtained from ESN and HIV⁺-P (both individuals from the SDC), HD and C. For CT only plasma samples were obtained with the purpose of being used as control group for ADCC assays. In all cases, blood samples were collected and centrifuged to separate plasma, which was stored at − 80 °C until use. For ADCC assays, plasma samples were first diluted (10-fold in RPMI medium) passed through a 0.2 μm-pore filter and heat-inactivated (1 h, 56 °C). PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation (GE Healthcare, UK) and cryopreserved for subsequent assays. PBMCs from one HIV negative donor were isolated, cryopreserved and used as effector cells in ADCC assay. Cells from the same donor were used in all assays to avoid bias from donor to donor. Plasma VL (branched-DNA, Versant HIV-1 RNA 3.0 assay; Siemens Healthcare, UK) and CD4⁺ T-cell count (flow cytometry double platform, BD FACSCanto; BD Biosciences, USA) were assessed in all subjects.

Ruiz M.J., Salido J., Abusamra L., Ghiglione Y., Cevallos C., Damilano G., Rodriguez A.M., Trifone C., Laufer N., Giavedoni L.D., Sued O., Salomón H., Gherardi M.M, & Turk G. (2017). Evaluation of Different Parameters of Humoral and Cellular Immune Responses in HIV Serodiscordant Heterosexual Couples: Humoral Response Potentially Implicated in Modulating Transmission Rates. EBioMedicine, 26, 25-37.

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PBMC Isolation and Characterization

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Blood samples were collected at enrollment on tubes with EDTA and centrifuged to separate plasma. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (Amersham, Sweden) and cryopreserved by standard procedures. Plasma VL (branched-DNA, Versant HIV-1 RNA 3.0 assay; Siemens Healthcare, UK) and CD4/CD8 counts (flow cytometry double platform, BD FACSCanto; BD Biosciences) were routinely determined in samples from all HIV infected patients and in samples from the HIV-neg donors enrolled at the Hospital General de Agudos “Dr. Juan A. Fernández”. Subsequent functional assays were performed according to sample availability, using only thawed cells with >95% viability after overnight rest in complete RPMI medium [RPMI-1640 (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 2 mM L-glutamine (Sigma-Aldrich, USA), 100 U/ml penicillin (Sigma-Aldrich, USA), 100 mg/ml streptomycin (Sigma-Aldrich, USA), and 10 mM HEPES (Gibco, USA)].

Caruso M.P., Falivene J., Holgado M.P., Zurita D.H., Laufer N., Castro C., Nico Á., Maeto C., Salido J., Pérez H., Salomón H., Cahn P., Sued O., Fink V., Turk G, & Gherardi M.M. (2019). Impact of HIV-ART on the restoration of Th17 and Treg cells in blood and female genital mucosa. Scientific Reports, 9, 1978.

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Characterization of PLWHA Immune Response

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To characterize PLWHA group, a blood sample (4 mL), after a 12-hr fasting period, was collected 48 hr before the exercise protocol. T CD4⁺ and T CD8⁺ lymphocytes were quantitated by flow cytometry, using FACSCalibur TM system (BD Biosciences, Franklin Lakes, NJ, USA). Viral load (bDNA) was measured by the VERSANT HIV-1 RNA 3.0 Assay (Siemens, Munich, Germany).

Deresz L.F., Karsten M., Corrêa I.F., Sonza A., Ikeda M.L., da Silva C.S, & Lago P.D. (2018). Functional capacity and ventilatory efficiency are preserved in well-controlled people living with human immunodeficiency virus/acquired immunodeficiency syndrome. Journal of Exercise Rehabilitation, 14(4), 680-687.

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Absolute CD4+ T Cell Counts and Viral Load

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Absolute CD4⁺ T cell counts were obtained using the MultiTest TruCount-kit and the MultiSet software on a FACSCalibur flow cytometer (BD Biosciences San Jose, CA). Plasma VL were measured according to the Brazilian Ministry of Health guidelines, with methodologies being updated overtime to improve sensitivity: Nuclisens HIV-1 RNA QT assay (Organon Teknika, Durham, NC, limit of detection: 80 copies/mL) from 1999 to 2007; the Versant HIV-1 3.0 RNA assay (bDNA 3.0, Siemens, Tarrytown, NY, limit of detection: 50 copies/mL) from 2007 to 2013; and the Abbott RealTime HIV-1 assay (Abbott Laboratories, Wiesbaden, Germany, limit of detection: 40 copies/mL) from 2013 to 2016.

de Azevedo S.S., Caetano D.G., Côrtes F.H., Teixeira S.L., dos Santos Silva K., Hoagland B., Grinsztejn B., Veloso V.G., Morgado M.G, & Bello G. (2017). Highly divergent patterns of genetic diversity and evolution in proviral quasispecies from HIV controllers. Retrovirology, 14, 29.

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CD4+, CD8+ T Cells and Viral Load Measurement

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Absolute CD4⁺ T and CD8⁺ T cell counts were obtained using the Tritest or MultiTest TruCount kit and the MultiSet software on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Plasma VL was measured using the Nuclisens HIV-1 RNA QT assay (Organon Teknika, Durham, NC, USA; limit of detection: 80 copies/mL) from 1999 to 2008, the Versant HIV-1 3.0 RNA assay (bDNA 3.0, Siemens, Tarrytown, NY, USA; limit of detection: 50 copies/mL) from 2008 to 2013, and the Abbott Real Time HIV-1 assay (Abbott Laboratories, Wiesbaden, Germany; limit of detection: 40 copies/mL) since 2013. When available, absolute CD4⁺ T and CD8⁺ T cell counts and VL data were collected from 1997 to 2017, depending on the HIC study entry.

Côrtes F.H., de Paula H.H., Bello G., Ribeiro-Alves M., de Azevedo S.S., Caetano D.G., Teixeira S.L., Hoagland B., Grinsztejn B., Veloso V.G., Guimarães M.L, & Morgado M.G. (2018). Plasmatic Levels of IL-18, IP-10, and Activated CD8+ T Cells Are Potential Biomarkers to Identify HIV-1 Elite Controllers With a True Functional Cure Profile. Frontiers in Immunology, 9, 1576.

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