Imputation results for rs2055272 were further validated by TaqMan genotyping (assay C_3025729_20, ThermoFisher Scientific) in 102 patients (98.03% concordance). Validation was performed by droplet digital PCR (ddPCR) approach using a QX200 Droplet Generator (BioRad) and the data was analyzed by QuantaSoft software (BioRad). Briefly, a ddPCR mastermix was prepared containing 11 μl 2× ddPCR Supermix (Bio-Rad), 1.1 μl 20× Taqman SNP Genotyping Assay (Applied Biosystems), and 7.9 μl nuclease-free water (Qiagen) per sample. The mastermix was prepared at room temperature and 20 μl was added to 2 μl (5 ng) of each DNA sample. Samples were loaded into individual wells of DG8TM cartridges (BioRad), and droplets were generated using a QX200 Droplet Generator (BioRad). For each sample, 40 μl of droplet mix was then transferred to a 96-well plate, and PCR was performed in a thermal cycler using the following cycling conditions: 95°C × 10 min; 40 cycles of [94°C × 30s, 60°C × 60s]; 98°C × 10s; 40°C × 10 min. The Bio-Rad QX200 Droplet Reader was then used to assess droplets as positive or negative based on fluorescence amplitude. The QuantaSoft software (BioRad) was used to analyze droplet data.
Qx200 droplet generator
Manufactured by Bio-Rad
Sourced in United States, Germany, Australia
The QX200 Droplet Generator is a lab equipment designed to generate uniform, monodisperse droplets for a variety of applications. It efficiently partitions samples into thousands of nanoliter-sized droplets, which can then be used for digital PCR, single-cell analysis, and other droplet-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Qx200 droplet reader, by Bio-Rad (225 mentions)
Quantasoft software, by Bio-Rad (84 mentions)
Ddpcr supermix for probe, by Bio-Rad (64 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (49 mentions)
C1000 touch thermal cycler, by Bio-Rad (48 mentions)
Ddpcr supermix for probes no dutp, by Bio-Rad (42 mentions)
T100 thermal cycler, by Bio-Rad (38 mentions)
Qx200 ddpcr evagreen supermix, by Bio-Rad (32 mentions)
Droplet generation oil for probe, by Bio-Rad (31 mentions)
Dg8 cartridge, by Bio-Rad (27 mentions)
Droplet generation oil, by Bio-Rad (25 mentions)
Ddpcr supermix, by Bio-Rad (23 mentions)
Px1 pcr plate sealer, by Bio-Rad (19 mentions)
Quantasoft analysis software, by Bio-Rad (14 mentions)
Qx200 droplet digital pcr ddpcr system, by Bio-Rad (11 mentions)
Quantasoft, by Bio-Rad (11 mentions)
Ddpcr evagreen supermix, by Bio-Rad (11 mentions)
Quantasoft v 1, by Bio-Rad (10 mentions)
Quantasoft analysis pro software, by Bio-Rad (9 mentions)
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
343 protocols using qx200 droplet generator
1
Validation of Genetic Variant Imputation
2
Confirming Mutations via ddPCR Analysis
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The mutations identified by WGS were further confirmed by Droplet Digital Polymerase Chain Reaction (ddPCR) technique using a QX200 Droplet Generator (BioRad) and the data was analyzed by QuantaSoft software (BioRad). Briefly, a ddPCR mastermix was prepared containing 11 μl 2X ddPCR Supermix (BioRad), 1.1 μl 20X TaqMan SNP Genotyping Assay (BioRad, ThermoFisher Scientific; Supplementary Table 6 ), and 7.9 μl nuclease-free water (Qiagen) per sample. The mastermix was prepared at room temperature and 20 μl was added to 2 μl (5 ng) of each DNA sample. Samples were loaded into individual wells of DG8TM cartridges (BioRad), and droplets were generated using a QX200 Droplet Generator (BioRad). For each sample, 40 μl of droplet mix was then transferred to a 96-well plate, and PCR was performed in a thermal cycler using the following cycling conditions: 95 °C × 10 min; 40 cycles of [94 °C × 30 s, 60 °C × 60 s]; 98 °C × 10 s; 40 C × 10 min. The BioRad QX200 Droplet Reader was then used to assess droplets as positive or negative based on fluorescence amplitude. The QuantaSoft software (BioRad) was used to analyze droplet data.
3
Droplet Digital PCR for mtDNA Quantification
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Droplet dPCR was conducted using the reference mtDNA and APOC3 nDNA assays indicated in Supplemental Table 7 . dPCR amplifications were analyzed as a simplex in a 24 μl reaction containing 1× dPCR Supermix for Probes (no dUTP; Bio-Rad), 250 nM of probe, 900 nM of each primer, 20U/μl Hind-III HF, and cellular DNA. For the APOC3 nuclear assay, 90 ng cellular DNA was added. For the reference mtDNA assay, 0.225 ng cellular DNA was added. Droplets were generated using a QX200 droplet generator (Bio-Rad), and PCR was performed on a C1000 Touch thermal cycler (Bio-Rad). Cycling conditions were as follows: one cycle of 95 ºC (2 ºC/s ramp) for 10 min, 45 cycles of 94 ºC (2 ºC/s ramp) for 10 s, 59.2 ºC (2 ºC/s ramp) for 30 s, 72 ºC (0.2 ºC/s ramp) for 1 min, one cycle of 98 ºC for 10 min, four ºC hold. Droplets were analyzed using a QX200 droplet reader (Bio-Rad), and QuantaSoft analysis software (Bio-Rad) was used to acquire and analyze data. mtDNA copy number was calculated by multiplying the concentration (copies/μl) of reference mtDNA–positive droplets by the dilution factor (400) and dividing by the concentration of APOC3-positive droplets.
4
Circulating Cell-Free DNA Quantification
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The copy number of cfDNA was determined using the QX200 droplet digital PCR (ddPCR) (Bio-Rad) system to detect tumor-associated BRAF V600E mutation, as previously described 50 (link). Briefly, the reaction mixture (supermix, primers/probe, cfDNA sample and water) were prepared according to the manufacturer's instruction. The QX200 Droplet Generator (Bio-Rad) partitions each 20 µL reaction mixture into more than 10,000 nanoliter-sized water-in-oil droplets for PCR amplification, which was performed using the following conditions: 1 cycle of 95°C for 10 min, 40 cycles of 94°C for 30 s and 55°C for 1 min, and 1 cycle of 98°C for 10 min. Following amplification, droplets from each sample were analysed individually on the QX200 Droplet Reader (Bio-Rad), where PCR-positive and PCR-negative droplets are counted to provide absolute quantification of target DNA in digital form. The DNA copy number per 20 µL reaction for the mutant and wild-type circulating DNA species was determined with Quantasoft software version 1.7.4 (Bio-Rad, Hercules, CA, USA) using a manual threshold setting. All samples with fewer than three positive mutant droplets were considered negative to improve specificity.
5
Quantifying Circulating Free DNA in Cancer
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The first blood sample was collected at the time of diagnosis before the start of treatment. Circulating free DNA (cfDNA) was quantified using the QX200 Droplet Digital PCR System (Bio-Rad Laboratories, Hercules, CA). For the EGFR multiplex assays, a final PCR mix volume of 20 μL was manually loaded into wells of a DG8 cartridge (Bio-Rad Laboratories) with 70 μL of Droplet Generation Oil for probes (Bio-Rad Laboratories). After droplet generation by the QX200 Droplet Generator (Bio-Rad Laboratories), 40 μL of the sample was transferred into a 96-well PCR plate and amplified with a C1000 Touch Thermal Cycler (Bio-Rad Laboratories), using the following thermal cycling conditions: 95 °C for 10 min, 40 cycles at 94 °C for 30 s, 55 °C for 1 min (2 °C/s), and 98 °C for 10 min, and a final cooling step to 12 °C (1 °C/s). The droplets were analyzed using the QX200 Droplet Reader (Bio-Rad Laboratories), and data were analyzed using QuantaSoft software version 1.7.4.0917 and QuantaSoft Analysis Pro software version 1.0.596 (Bio-Rad Laboratories). Thresholds were placed manually, and the fractions of positive and negative droplets were used to calculate the concentration and fractional abundance of target DNA sequences with their 95% Poisson-based confidence intervals (CI). EGFRm primers and probes were based on previous studies [27 (link), 28 (link)].
6
Quantification of Cell-Associated SIV RNA and DNA
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Total cell-associated SIV RNA was quantified using RT-ddPCR using 100 ng of RNA, a 1-Step RT-ddPCR Advanced kit for probes (Bio-Rad; cat. no. 1864022), and the same set of primers and probe used for SIV plasma viral load quantification. ddPCR was carried out on a Bio-Rad QX200 AutoDG digital droplet PCR system. In brief, 22 μl of reaction mix was used for droplet generation using a QX200 droplet generator, the ddPCR plate having the emulsified samples was heat sealed with foil (Bio-Rad; cat. no. 181-4040), and amplification occurred in a C1000 Touch thermal cycler (Bio-Rad, CA, USA). After thermal cycling, ddPCR plates were transferred to the QX200 droplet reader (Bio-Rad) for droplet count and fluorescence measurement. Positive droplets with amplified products were separated from negative droplets without target amplicon by applying a fluorescence amplitude threshold, and the absolute quantity of RNA per sample (copies/µl) was determined using QuantaSoft software. For quantification of cell-associated SIV DNA, the same methodology was used as described above without using the reverse transcription step and with 2× ddPCR Supermix for probes (no dUTP) (Bio-Rad; cat. no. 1863024) instead of the 1-Step RT-ddPCR Advanced kit.
7
Digital Droplet PCR for DDOST RNA Editing
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DDOST 558C>U RNA-editing assays were performed as described previously with assistance from the MSKCC Integrated Genomics Operation28 (link). Total RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. After extraction, the RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). cDNA (20 ng) along with primers purchased from Bio-Rad (10031279 and 10031276) for the target DDOST558C>U amplification were mixed in PCR reactions in a total volume of 25 μl. Then, 20 μl of the reactions were mixed with 70 μl of Droplet Generation Oil for Probes (Bio-Rad) and loaded into a DG8 cartridge (Bio-Rad). A QX200 Droplet Generator (Bio-Rad) was used to make the droplets, which were transferred to a 96-well plate and the following PCR reaction was then run: 5 min at 95 °C; 40 cycles of 94 °C for 30s and 53 °C for 1 min; and finally 98 °C for 10 min. The QX200 Droplet Reader (Bio-Rad) was then used to analyse the droplets for fluorescence measurement of the fluorescein amidite (FAM) and hexachloro-fluorescein (HEX) probes. The data were analysed using the QuantaSoft analysis software (Bio-Rad) and gating was performed on the basis of positive and negative DNA oligonucleotide controls.
8
Droplet Digital PCR for Detecting Mutations
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ddPCR assays were performed with the Prime-PCR™ ddPCR™ Mutation Detection Assay Kit (Bio-Rad, Hercules, CA, USA) using an amplicon of 57 nt. DNA from the SW480 cell line was used as a positive control and DNA from leukocytes of a healthy donor served as a negative control. Background from water added to the reaction mixture instead of DNA was analyzed. This study was performed on a QX200 Droplet Digital PCR System (Bio-Rad), consisting of a C1000 Touch Thermalcycler, a QX200 Droplet Generator, and a QX200 Droplet Reader. The PCR reaction mixture (20 µL) contained 10 µL of ddPCR Supermix (no dUTP) for probes, 1 µL of each primer/probe mix (target and reference, labeled with HEX and FAM fluorophores, respectively), and 8 µL of plasma-extracted DNA. A total amount of 130 ng of DNA was added per well in case of positive and negative controls and in DNA extracted from tumors. The thermal cycling started with 10 min at 95 °C, followed by 40 cycles of 94 °C for 30 s and 55 °C for 60 s. Results were analyzed using Quantasoft v.1.7 software (Bio-Rad) and reported as copies per mL of plasma or % of mutant DNA in the tumor. Four replicates of each sample were analyzed.
9
Droplet Digital PCR Protocol for Quantification
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Droplet digital PCR was performed as described [19 (link)]. Briefly, each ddPCR assay mixture (20 μl) was loaded into a disposable droplet generator cartridge (Bio-Rad). Then, 70 μl of droplet generation oil for probes (Bio-Rad) was loaded into each of the eight oil wells. The cartridge was then placed inside the QX200 droplet generator (Bio-Rad). When droplet generation was completed, the droplets were transferred to a 96-well PCR plate (Eppendorf) using a Rainin multichannel pipet. The plate was heat-sealed with foil and placed in a conventional thermal cycler. Thermal cycling conditions were: 95°C for 5 min, then 40 cycles of 95°C for 30 s and 58°C for 1 min (ramping rate reduced to 2%), and three final steps at 4°C for 5 min, 90°C for 5 min and a 4°C indefinite hold. A no template control (NTC) and a negative control for each reverse transcription reaction (RT-neg) were included in every assay. Cel-miR-39 assay was performed to monitor RT reaction efficacy.
10
Quantitative DNA Mutation Detection via ddPCR
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