Droplet digital pcr ddpcr system

Manufactured by Bio-Rad

Sourced in United States

The Droplet Digital PCR (ddPCR) system is a highly sensitive and precise molecular detection and quantification platform. It utilizes water-oil emulsion droplet technology to partition samples into thousands of individual reaction compartments, enabling absolute quantification of target nucleic acid sequences with high precision and sensitivity.

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Lab products found in correlation

Geneassist copy number assay workflow builder, by Thermo Fisher Scientific (2 mentions) Taqman target probe fam, by Thermo Fisher Scientific (2 mentions) Taqman reference probe vic, by Thermo Fisher Scientific (2 mentions) Superscript 3 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions) Taqman copy number assay probes, by Bio-Rad (1 mentions) Rneasy kit, by Qiagen (1 mentions)

Spelling variants of this product

Droplet digital PCR (70 citations) QX200 droplet digital PCR (ddPCR) system (59 citations) Droplet digital PCR (ddPCR; (19 citations) DdPCR system (14 citations) QX100 droplet digital PCR (ddPCR) system (9 citations) QX200 AutoDG droplet digital PCR (ddPCR) system (2 citations)

5 protocols using droplet digital pcr ddpcr system

Comprehensive EGFR Mutation Profiling

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The ddEGFR test was performed in a 20 μL volume containing 3.3 ng (1,000 GE)/reaction of template DNA on a Droplet Digital™ PCR (ddPCR) system (Bio-Rad, Hercules, CA, USA). The ddPCR assay was conducted as described previously³³. Thresholds for detection were set manually based on results from non-template control wells and negative control wells containing wild-type gDNA (Promega). PCR amplification for the cobas EGFR test (Roche Molecular Systems Inc., Branchburg, NJ, USA) was performed on a cobas z 480 Analyzer. The cobas EGFR test requires 150 ng total input DNA. Both mutation tests were analyzed in a double-blind fashion, and the results were matched after analysis.
For mutation screening of EGFR exons 18, 19, 20, and 21 by 2× bidirectional Sanger sequencing, regions of interest were amplified by PCR, and the amplified samples were processed at an independent laboratory (Macrogen, Seoul, Korea) using a validated protocol. Sanger sequencing results were cross-checked and interpreted by a pathologist (Y.L.C.).

Kim S.S., Choi H.J., Kim J.J., Kim M.S., Lee I.S., Byun B., Jia L., Oh M.R., Moon Y., Park S., Choi J.S., Chae S.W., Nam B.H., Kim J.S., Kim J., Min B.S., Lee J.S., Won J.K., Cho S.Y., Choi Y.L, & Shin Y.K. (2018). Droplet digital PCR-based EGFR mutation detection with an internal quality control index to determine the quality of DNA. Scientific Reports, 8, 543.

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Droplet Digital PCR for Copy Number Assay

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Copy number assays were performed using the Droplet Digital PCR (ddPCR) system (Bio‐Rad Laboratories, Inc.). The GeneAssist Copy Number Assay Workflow Builder (Thermo Fischer) was used to design TaqMan assays on Chr15, SNHG14 (Hs05375107_cn) with FAM dye. TaqMan Copy Number Reference Assay, human, RNase P with VIC dye was used as a reference assay. A total of 22 μL reaction mix was prepared, containing 3.5 μL of template DNA (20 ng/μL) without restriction digestion, 10 μL of 2× ddPCR supermix for probes (no UDP) (Bio‐Rad Laboratories Inc.), 1 μL each 20× TaqMan target probe (FAM) and 20× TaqMan reference probe (VIC) (Applied Biosystems), and 6.5 μL of RNase‐/DNase‐free water (see the Supporting Information section for details). All reactions were prepared in triplicates with one negative control. The reaction mixtures were partitioned using the QX200 Droplet Generator and then transferred to a 96‐well plate and amplified using the C1000 Touch thermal cycler, following the manufacturer's protocol. The samples were then read using the QX200 Droplet Reader. Data acquisition and analysis were performed using QuantaSoft Version 1.7.4.0917, and the Poisson algorithm was used to determine the concentrations of the targets as copies/μL.

Mim R.A., Soorajkumar A., Kosaji N., Rahman M.M., Sarker S., Karuvantevida N., Eshaque T.B., Rahaman M.A., Islam A., Chowdhury M.S., Shams N., Uddin K.M., Akter H, & Uddin M. (2024). Expanding deep phenotypic spectrum associated with atypical pathogenic structural variations overlapping 15q11–q13 imprinting region. Brain and Behavior, 14(4), e3437.

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Vector Copy Number Quantification by ddPCR

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The vector copy number was determined using a Droplet Digital PCR (ddPCR) system (Bio-Rad, Hercules, CA, USA) with specific primers and probes for HIV and albumin sequences.

Moirangthem R.D., Ma K., Lizot S., Cordesse A., Olivré J., de Chappedelaine C., Joshi A., Cieslak A., Tchen J., Cagnard N., Asnafi V., Rausell A., Simons L., Zuber J., Taghon T., Staal F.J., Pflumio F., Six E., Cavazzana M., Lagresle-Peyrou C., Soheili T, & André I. (2021). A DL-4- and TNFα-based culture system to generate high numbers of nonmodified or genetically modified immunotherapeutic human T-lymphoid progenitors. Cellular and Molecular Immunology, 18(7), 1662-1676.

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Quantitative Measurement of cDNA Expression

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RNA was extracted using the Rneasy kit (Qiagen, Hilden, Germany). cDNA was then generated using the Superscript III Reverse Transcriptase kit (ThermoFisher). Differential levels of cDNA expression were measured using the droplet digital PCR (ddPCR) system (Bio-Rad) and TaqMan copy number assay probes or primers (Table S4). Briefly, cDNA and 6-carboxyfluorescein probes or primers were distributed into 10,000–20,000 droplets. The nucleic acids were then PCR-amplified in a thermal cycler and read (as the number of positive and negative droplets) with a QX200 ddPCR system. The results were normalized as follows: the IgG IP results were subtracted from the RACK1 RNA IP results for each gene. The results were then normalized against 18S rRNA gene expression. For GFP RNA IP (TRAP), results were normalized against the 18S rRNA.

Oudart M., Avila-Gutierrez K., Moch C., Dossi E., Milior G., Boulay A.C., Gaudey M., Moulard J., Lombard B., Loew D., Bemelmans A.P., Rouach N., Chapat C, & Cohen-Salmon M. (2023). The ribosome-associated protein RACK1 represses Kir4.1 translation in astrocytes and influences neuronal activity. Cell Reports, 42(5), 112456.

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Precise Copy Number Quantification via ddPCR

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Copy number assays were performed using the Droplet Digital PCR (ddPCR) System (Bio-Rad Laboratories, Inc.). The GeneAssist™ Copy Number Assay Workflow Builder (Thermo Fischer, United States) was used to design TaqMan assays on Chr11, PKNOX2 (Hs03289418_cn), Chr15, SNHG14 (Hs05375107_cn), and Chr21, TSPEAR (Hs02835328_cn) with FAM dye. TaqMan™ Copy Number Reference Assay, human, RNase P with VIC dye was used as a reference assay. A total of 22 µL reaction mix was prepared, containing 3.5 µL of template DNA (20 ng/μL) without restriction digestion, 10 µL of 2X ddPCR supermix for probes (No UDP) (Bio-RAD Laboratories Inc.), 1 µL each 20X TaqMan target probe (FAM) and 20X TaqMan reference probe (VIC) (Applied Biosystems, United States), and 6.5 µL of RNase-/DNase-free water. All reactions were prepared in triplicates with one negative control. The reaction mixtures were partitioned using the QX200 Droplet Generator™ and then transferred to a 96-well plate and amplified using the C1000 Touch thermal cycler, as per the manufacturer’s protocol. Then, the samples were read using the QX200 Droplet Reader™. Data acquisition and analysis were performed using QuantaSoft Version 1.7.4.0917, and the Poisson algorithm was used to determine the concentrations of the targets as copies/μL.

Akter H., Rahman M.M., Sarker S., Basiruzzaman M., Islam M.M., Rahaman M.A., Rahaman M.A., Eshaque T.B., Dity N.J., Sarker S., Amin M.R., Hossain M.M., Lopa M., Jahan N., Hossain S., Islam A., Mondol A., Faruk M.O., Saha N., Kundu G.K., Kanta S.I., Kazal R.K., Fatema K., Rahman M.A., Hasan M., Hossain Mollah M.A., Hosen M.I., Karuvantevida N., Begum G., Zehra B., Nassir N., Nabi A.H., Uddin K.M, & Uddin M. (2023). Construction of copy number variation landscape and characterization of associated genes in a Bangladeshi cohort of neurodevelopmental disorders. Frontiers in Genetics, 14, 955631.

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