Total RNA was isolated from 1×106 fresh peripheral blood mononuclear cells (PBMCs) of healthy donors using High Pure RNA Isolation Kit (Roche). BLK expression was measured by relative quantification using QuantStudio™ 3D Digital PCR System with probes Hs01017452_m1 for BLK (FAM), and Hs001003268 for HPRT1 (VIC) normalizer using cDNA equivalent to 10 ng and 20 ng RNA. Fluorescence in QuantStudio™ 3D Digital PCR 20K Chips was measured by QuantStudio™ 3D Digital PCR Instrument and data were analyzed by QuantStudio™ 3D AnalysisSuite™ Cloud Software (Life Technologies). As BLK mRNA expression in CD19+ B cells is at least 30-fold higher than in other BLK-expressing leukocytes (13 (link)), we have considered that the measured BLK expression comes mainly from B lymphocytes. Thus, the quantity of BLK mRNA measured by dRT-PCR was estimated using the formula: BLK expression in B lymphocytes = 100* BLK expression in PBMC / % CD19+ cells in PBMCs. The frequency of CD19+ B cells in the PBMCs was determined by FACS analysis.
Quantstudio 3d digital pcr instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio 3D Digital PCR Instrument is a laboratory equipment designed for digital PCR analysis. It provides high-precision quantification of nucleic acid targets by partitioning samples into thousands of individual reaction chambers and performing real-time PCR on each partition. The instrument's core function is to enable sensitive and accurate quantification of DNA and RNA molecules.
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Lab products found in correlation
Quantstudio 3d digital pcr system, by Thermo Fisher Scientific (8 mentions)
Quantstudio 3d digital pcr master mix, by Thermo Fisher Scientific (7 mentions)
Quantstudio 3d analysissuite cloud software, by Thermo Fisher Scientific (5 mentions)
Proflex pcr system, by Thermo Fisher Scientific (5 mentions)
Proflex 2x flat pcr system, by Thermo Fisher Scientific (5 mentions)
Quantstudio 3d digital pcr chip, by Thermo Fisher Scientific (4 mentions)
Quantstudio 3d digital pcr master mix v2, by Thermo Fisher Scientific (4 mentions)
Quantstudio 3d digital polymerase chain reaction system, by Thermo Fisher Scientific (2 mentions)
Quantstudio analysissuite software, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3d analysis suite, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3d digital pcr chip loader, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3d analysissuite software, by Thermo Fisher Scientific (2 mentions)
Sybr green 1 dye, by Thermo Fisher Scientific (2 mentions)
Plasma serum circulating and exosomal rna purification mini kit, by Thermo Fisher Scientific (2 mentions)
High pure rna isolation kit, by Roche (1 mentions)
Biorobot ez1, by Qiagen (1 mentions)
Ez1 dna tissue kit, by Qiagen (1 mentions)
Ez1 bacteria card, by Qiagen (1 mentions)
Iprep purification instrument, by Thermo Fisher Scientific (1 mentions)
Limulus amebocyte lysate assay, by Cambrex (1 mentions)
24 protocols using quantstudio 3d digital pcr instrument
1
Quantifying BLK Expression in B Cells
2
Bacterial DNA Quantification in PD Effluent
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A 20-mL specimen of PD effluent was collected on randomization (i.e. 5 days before antibiotic completion, according to the ISPD guideline) for the measurement of bacterial DNA fragment levels. For the extended group, a second PD effluent sample was collected 5 days before the completion of the extended treatment. DNA was extracted using the EZ1 DNA tissue kit and BioRobot EZ1 with the EZ1 bacteria card (Qiagen, Germantown, MD, USA), according to the manufacturer’s instructions. Purified DNA was eluted in 50 µL of elution buffer before amplification. The bacterial DNA fragment level in PD effluent was measured by the QuantStudio 3D Digital Polymerase Chain Reaction (PCR) System (Life Technologies, Carlsbad, CA, USA). Briefly, the PCR mixture was prepared and loaded into the chip according to the manufacturer’s protocol. PCR amplification was performed by the ProFlex µPCR system (Life Technologies). The result was captured by the QuantStudio 3D Digital PCR Instrument and analyzed by the QuantStudio AnalysisSuite Software (both from Life Technologies).
3
Quantitative Assessment of Transduced CD8 T Cells
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Genomic DNA was isolated from transduced CD8 T cells with the iPrep Purification Instrument (Thermo fisher scientific) and qPCR analysis was performed using ABI Taqman technology, with a modified version of the previously described assay designed to detect the integrated CD4-zeta sequence in genomic DNA (gDNA) [22 (link)]. To determine copy number per unit DNA, a standard curve was generated consisting of 5 to 106 plasmid copies spiked into 200 ng nontransduced control gDNA. The plasmid copy number in the standard curve was verified using digital qPCR with the same CD4-z primer/probe set, and performed on a QuantStudio 3D digital PCR instrument (Life Technologies). Each data-point was evaluated in triplicate with a positive Ct value and % CV less than 0.95% for all quantifiable values. To control for the quantity of interrogated DNA, a parallel amplification reaction was performed using 10 ng gDNA and a primer/probe set specific for a non-transcribed genomic sequence upstream of the CDKN1A (p21) gene as previously described [72 (link)]. These amplification reactions generated a correction factor to adjust for calculated versus actual DNA input. Copies of transgene per cell were calculated according to the formula: [Average copies of transgene(from qPCR)x gDNA input Correction Factor/Input gDNA(ng)]x 0.0063 ng gDNA/cell.
4
SARS-CoV-2 sgRNA Detection by Digital PCR
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The presence of sg-RNA was assessed by specific retrotranscription (see [12 (link)]) and digital PCR (dPCR) in 122 longitudinal RNA samples from a sub-cohort of 36 individuals characterized by age, sex distribution and basal viral load compared to those of the whole cohort. The resulting cDNAs were then tested by means of chip-based dPCR using TaqMan assays specific for N and E genes on QuantStudio 3D Digital PCR System (Life Technologies, Carlsbad, CA, USA). In particular, 6 µL of each cDNA was combined with QuantStudio™ 3D Digital PCR Master Mix (Life Technologies) and the TaqMan assay specific for N or E gene and, then, loaded on QuantStudio™ 3D Digital PCR Chips according to manufacturer’s instructions. Each sample was tested in duplicate on two different chips. Loaded samples were then amplified on ProFlex PCR system (Life Technologies) and the end-point fluorescent data were analyzed by QuantStudio™ 3D Digital PCR Instrument (Life Technologies) and the QuantStudio™ 3D AnalysisSuite™ (Life Technologies).
5
Quantifying Bacterial Burden in Plasma
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Circulating bacterial fragment was represented by plasma endotoxin and bacterial DNA levels. Plasma endotoxin level was measured by a commercially available Limulus Amebocyte Lysate assay (Cambrex, Verviers, Belgium) as described previously [21 (link)]. All samples were diluted to 20% with endotoxin-free water and then heated to 70°C for 10 min to inactivate plasma proteins. The detection limit of the assay was 0.01 EU/mL. Plasma bacterial DNA level was measured by the QuantStudio 3D Digital Polymerase Chain Reaction (PCR) System (Life Technologies, Carlsbad, CA, USA) as described previously [22 (link)]. In essence, PCR amplification was performed by the ProFlex µPCR system, the result captured by the QuantStudio 3D Digital PCR Instrument, and analyzed by the QuantStudio Analysis Suite Software (all from Life Technologies).
6
Digital PCR Quantification of Genetic Markers
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Genomic DNA was extracted from uncultured CVS (approximately 1–5 mg) or AF (approximately 4–8 mL) using MagCore following proteinase K treatment and suspended in a final volume of 60 μL, according to the manufacturer’s protocol (RBC Bioscience, Taipei, Taiwan). The extracted DNA was measured by NanoDrop spectrophotometer (Life Technology, Carlsbad, CA, USA).
The digital PCR amplification was performed using a QuantStudio 3D Digital PCR System (Life Technology, Carlsbad, CA, USA) in a total reaction volume of 15 μL containing 7.5 μL QuantStudio 3D Digital PCR Master Mix (Life Technology, Carlsbad, CA, USA), 0.75 μL of the custom TaqMan Assays (Life Technology, Carlsbad, CA, USA), and 1 to 10 ng DNA template diluted in 6.75 μL. The primer and probe sequences and target combinations in duplex reactions are described elsewhere [16 (link)]. The reaction mixture was loaded onto a chip using QuantStudio 3D Digital PCR Chip Loader (Life Technology, Carlsbad, CA, USA). Thermal cycling was performed according to the manufacturer’s instructions: 96 °C for 10 min, 39 cycles of 60 °C for 2 min, 98 °C for 30 s, and a final extension at 60 °C for 2 min. The fluorescence signals of VIC and FAM were measured using QuantStudio 3D Digital PCR Instrument (Life Technology, Carlsbad, CA, USA).
The digital PCR amplification was performed using a QuantStudio 3D Digital PCR System (Life Technology, Carlsbad, CA, USA) in a total reaction volume of 15 μL containing 7.5 μL QuantStudio 3D Digital PCR Master Mix (Life Technology, Carlsbad, CA, USA), 0.75 μL of the custom TaqMan Assays (Life Technology, Carlsbad, CA, USA), and 1 to 10 ng DNA template diluted in 6.75 μL. The primer and probe sequences and target combinations in duplex reactions are described elsewhere [16 (link)]. The reaction mixture was loaded onto a chip using QuantStudio 3D Digital PCR Chip Loader (Life Technology, Carlsbad, CA, USA). Thermal cycling was performed according to the manufacturer’s instructions: 96 °C for 10 min, 39 cycles of 60 °C for 2 min, 98 °C for 30 s, and a final extension at 60 °C for 2 min. The fluorescence signals of VIC and FAM were measured using QuantStudio 3D Digital PCR Instrument (Life Technology, Carlsbad, CA, USA).
7
Digital PCR Detection of Oncogenic Mutations
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3D Digital PCR analysis was performed on QuantStudio™ 3D Digital PCR System (Thermo Scientific, Inc., Waltham, MA, USA) using primers and probes for detection of BRAFV600E, TERTC228T, and TERTC250T (Thermo Fisher Scientific). The final 14.5 μL of reaction mixture contained 8.7 μL QuantStudio™ 3D Digital PCR Master Mix, 0.43 μL of primer/probe mix, 10 ng of DNA. The mixture was loaded into the QuantStudio™ 3D Digital PCR Chip. For BRAFV600E analysis chips were run on a ProFlex 2 × flat PCR System (Thermo Fisher) cycled with the following conditions: 1 cycle at 50 °C for 2 min; 1 cycle at 95 °C for 10 min; 45 cycles at 60 °C for 1 min and 95 °C for 15 s; 1 cycle at 60 °C for 1 min. For TERT promoter mutations, chips were run with the following conditions: 1 cycle at 50 °C for 2 min; 1 cycle at 95 °C for 10 min; 54 cycles at 55 °C for 1 min and 95 °C for 15 s; 1 cycle at 60 °C for 1 min. End point fluorescence data were collected on the QuantStudio™ 3D Digital PCR instrument and analyzed using the QuantStudio 3D AnalysisSuite software (Thermo Scientific, Inc., Waltham, MA, USA).
8
miRNA Expression Quantification by qRT-PCR
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In the validation phase of the study, cDNA synthesis was performed by TaqManTM Advanced miRNA cDNA Synthesis kit followed by qRT-PCR using TaqManTM Fast Advanced Master Mix with individual TaqMan Advanced miRNA assays (all ThermoFisher Scientific, Waltham, MA, USA) on the QuantStudio™ 3D Digital PCR Instrument (ThermoFisher Scientific, Waltham, MA, USA). All reactions were held according to manufacturer’s protocol.
9
Digital PCR Quantification Protocol
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Reaction mixtures were prepared by combining genomic DNA, RNase P assay, GLA assay, and QuantStudio™ 3D Digital PCR Master Mix v2 (PN A26358), according to the manufacturer’s instructions. Each reaction mixture was loaded onto a QuantStudio 3D Digital PCR Chip v2 (PN 100027736).
For all experiments, chips were run on a ProFlex™ 2 x flat PCR System (Applied Biosystems™) cycled with the following conditions: 10 min at 96.0 degree C; then 39 cycles at 60.0 degree C for 2 min, 30 s at 98.0 degree C; and a final elongation step of 2 min at 60 degree C. End-point fluorescence data were collected on the QuantStudio 3D Digital PCR instrument and analyzed using the QuantStudio 3D AnalysisSuite software (Thermo Fisher Scientific).
For all experiments, chips were run on a ProFlex™ 2 x flat PCR System (Applied Biosystems™) cycled with the following conditions: 10 min at 96.0 degree C; then 39 cycles at 60.0 degree C for 2 min, 30 s at 98.0 degree C; and a final elongation step of 2 min at 60 degree C. End-point fluorescence data were collected on the QuantStudio 3D Digital PCR instrument and analyzed using the QuantStudio 3D AnalysisSuite software (Thermo Fisher Scientific).
10
Digital RT-PCR for GSDMB Isoform Quantification
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To measure GSDMB Δ5–8 isoform levels and total GSDMB transcript, digital RT-PCR reactions were performed on a QuantStudio 3D Digital PCR System (Thermo Fisher Scientific) using the QuantStudio 3D Digital PCR Master Mix (Thermo Fisher Scientific) and 1 μL of cDNA as template. Custom TaqMan assays were designed to amplify GSDMB Δ5–8 isoform and total GSDMB isoforms. The sequences of primers and probes used in digital RT-PCR assays are listed in Supplementary Table S1 .
Each reaction mixture was loaded onto a QuantStudio 3D Digital PCR Chip (Thermo Fisher Scientific) and cycled for 40 cycles using standard conditions. End-point fluorescence data were analyzed through the QuantStudio 3D Digital PCR Instrument and the QuantStudio 3D Analysis Suite (Thermo Fisher Scientific), according to the manufacturer’s instructions.
Each reaction mixture was loaded onto a QuantStudio 3D Digital PCR Chip (Thermo Fisher Scientific) and cycled for 40 cycles using standard conditions. End-point fluorescence data were analyzed through the QuantStudio 3D Digital PCR Instrument and the QuantStudio 3D Analysis Suite (Thermo Fisher Scientific), according to the manufacturer’s instructions.
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