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Qx200 ddpcr instrument

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The QX200 ddPCR instrument is a digital PCR system designed for precise and accurate quantification of nucleic acids. The instrument partitions samples into thousands of nanoliter-sized droplets, allowing for the sensitive detection and quantification of target molecules. The core function of the QX200 ddPCR is to perform droplet digital PCR (ddPCR) analysis.

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Lab products found in correlation

High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions) Quantasoft v 1, by Bio-Rad (1 mentions) Allprep dna rna kit, by Qiagen (1 mentions) Allprep mini kit, by Qiagen (1 mentions) Allprep dna rna mini kit, by Qiagen (1 mentions) Qx200 droplet generator, by Bio-Rad (1 mentions) 3iabkfq, by Integrated DNA Technologies (1 mentions)

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QX200 (107 citations) DdPCR QX200 (12 citations) QX200 instrument (11 citations)

6 protocols using qx200 ddpcr instrument

1

Digital PCR Verification of Variant Blend

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To verify the expected concentration of a 5% variant blend of HD500 added into control cfDNA (Figure 4b; also see Reference standards for noninvasive tumor genotyping, above), we separately quantified 3 mutant alleles known to be present in the mixture: EGFR, KRAS, and BRAF on a Bio-Rad QX200 ddPCR instrument as previously described46 (link), using reagents, primers and probes obtained from Bio-Rad. We utilized previously validated PrimePCR assays for mutation quantification, where wild-type and mutant assays employed HEX and FAM labels, respectively. For EGFR L858R, wild-type and mutant alleles were profiled within a 73bp amplicon annealed at 56°C, using the corresponding assays (dHsaCP2000021/dHsaCP200002122). For KRAS G13D, wild-type and mutant alleles were profiled within a 57bp amplicon annealed at 53°C, using the corresponding assays (dHsaCP2500598/dHsaCP2500599). For BRAF V600E, wild-type and mutant alleles were profiled within a 91bp amplicon annealed at 56°C using the corresponding assays (dHsaCP2000027/dHsaCP2000028). PCR conditions employed 95°C × 10 min (1 cycle), 40 cycles of 94°C × 30 sec and assay-specific annealing temperature × 1 min, and 4°C hold. A 2°C per second ramping rate was used for all steps.
Newman A.M., Lovejoy A.F., Klass D.M., Kurtz D.M., Chabon J.J., Scherer F., Stehr H., Liu C.L., Bratman S.V., Say C., Zhou L., Carter J.N., West R.B., Sledge G.W., Shrager J.B., Loo BW J.r., Neal J.W., Wakelee H.A., Diehn M, & Alizadeh A.A. (2016). Integrated digital error suppression for improved detection of circulating tumor DNA. Nature biotechnology, 34(5), 547-555.
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2

Quantifying HIV DNA and RNA in PBMCs

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Peripheral blood mononuclear cells (PBMCs) were isolated and simultaneously analyzed for total proviral HIV DNA and CA-HIV RNA. Total proviral DNA was purified using AllPrep DNA/RNA kit (Qiagen) according to the manufacturer’s protocol. Quantitation of viral DNA was performed by droplet digital polymerase chain reaction (ddPCR) using a Bio-Rad QX200 ddPCR instrument. Following PCR amplification, a flow cytometry analysis was used to determine the fraction of PCR-positive droplets in the original sample. This data was then analyzed using Poisson statistics, implemented in Bio-Rad QuantaSoft v. 1.7.4, to determine the target DNA template concentration in the original sample. Total CA-HIV RNA was also extracted from the PBMCs using the AllPrep Mini Kit from Qiagen (catalog #80204). The total CA-HIV RNA was quantitated using the Superscript III Platinum One Step qRT-PCR Kit Catalog #11732–088 with primers and probe specific for the long terminal repeat sequence.
Oni O., Glynn T.R., Antoni M.H., Jemison D., Rodriguez A., Sharkey M., Salinas J., Stevenson M, & Carrico A.W. (2019). Post-Traumatic Stress Disorder, Cocaine Use, and HIV Persistence. International journal of behavioral medicine, 26(5), 542-550.
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3

Digital PCR Verification of Variant Blend

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
To verify the expected concentration of a 5% variant blend of HD500 added into control cfDNA (Figure 4b; also see Reference standards for noninvasive tumor genotyping, above), we separately quantified 3 mutant alleles known to be present in the mixture: EGFR, KRAS, and BRAF on a Bio-Rad QX200 ddPCR instrument as previously described46 (link), using reagents, primers and probes obtained from Bio-Rad. We utilized previously validated PrimePCR assays for mutation quantification, where wild-type and mutant assays employed HEX and FAM labels, respectively. For EGFR L858R, wild-type and mutant alleles were profiled within a 73bp amplicon annealed at 56°C, using the corresponding assays (dHsaCP2000021/dHsaCP200002122). For KRAS G13D, wild-type and mutant alleles were profiled within a 57bp amplicon annealed at 53°C, using the corresponding assays (dHsaCP2500598/dHsaCP2500599). For BRAF V600E, wild-type and mutant alleles were profiled within a 91bp amplicon annealed at 56°C using the corresponding assays (dHsaCP2000027/dHsaCP2000028). PCR conditions employed 95°C × 10 min (1 cycle), 40 cycles of 94°C × 30 sec and assay-specific annealing temperature × 1 min, and 4°C hold. A 2°C per second ramping rate was used for all steps.
Newman A.M., Lovejoy A.F., Klass D.M., Kurtz D.M., Chabon J.J., Scherer F., Stehr H., Liu C.L., Bratman S.V., Say C., Zhou L., Carter J.N., West R.B., Sledge G.W., Shrager J.B., Loo BW J.r., Neal J.W., Wakelee H.A., Diehn M, & Alizadeh A.A. (2016). Integrated digital error suppression for improved detection of circulating tumor DNA. Nature biotechnology, 34(5), 547-555.
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4

Quantitative Analysis of Gene Expression

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DNA and RNA were extracted from various tissues using the AllPrep DNA/RNA Mini Kit (QIAGEN; cat. no. 80204). RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific; cat no. 4368814). Taqman-based multiplexing droplet digital PCR (ddPCR) was performed using a QX200 ddPCR instrument (Bio-Rad). For both vector genome analysis and transgene expression analysis, a customized Taqman reagent targeting the codon-optimized human ASPA cDNA was used (Thermo Fisher Scientific; Assay ID APNKRZW). We used the Taqman reagents targeting mouse Tfrc genomic sequence (Invitrogen; cat. no. 4458367) and mouse Gusb cDNA sequence (Invitrogen; Assay ID Mm01197698_m1) as reference for vector genome analysis and transgene expression analysis, respectively.
Wang D., Li S., Gessler D.J., Xie J., Zhong L., Li J., Tran K., Van Vliet K., Ren L., Su Q., He R., Goetzmann J.E., Flotte T.R., Agbandje-McKenna M, & Gao G. (2018). A Rationally Engineered Capsid Variant of AAV9 for Systemic CNS-Directed and Peripheral Tissue-Detargeted Gene Delivery in Neonates. Molecular Therapy. Methods & Clinical Development, 9, 234-246.
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5

Quantifying HIV DNA by Digital Droplet PCR

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Quantitation of viral DNA was performed on extracted leukocyte DNA by digital droplet PCR (ddPCR) using a Bio-Rad QX200 ddPCR instrument (Bio-Rad Laboratories, Inc., Hercules, CA). Primers and probe bind to the LTR sequence with an amplicon length of 185 base pairs and are based on the HIVLAI clone (Genbank accession number K02013). The forward primer sequence is LAI8981ctg cat ccg gag tac ttc aag aac tg, the reverse primer sequence is LAI9166tcc cag gct caa atc tgg tct a and that of the fluorogenic reporter probe is LAI908156-FAM agt ggc gag /ZEN/ccc tca gat gct gc 3IABkFQ (Integrated DNA Technologies). To perform ddPCR, the DNA target, fluorescently-labeled probe, and the ingredients for PCR reaction were partitioned into 20,000 droplets by the QX200 Droplet Generator (BioRad). PCR amplification of the template molecules occurred in each individual droplet. Following PCR amplification, each droplet was analyzed for fluorescent signal in the QX200 droplet reader to determine the fraction of PCR-positive droplets in the original sample. The dynamic range of detection for ddPCR is from 1 to 1Χ105 copies (Bizouarn, 2014 ). The single-copy human CCR5 gene was quantified to measure the number of cell equivalents in DNA samples for standardization purposes (Sharkey et al., 2000 ).
Grosgebauer K., Salinas J., Roach M., Pallikkuth S., Dilworth S.E., Pahwa S., Koru-Sengul T., Stevenson M, & Carrico A. (2018). Psychosocial Correlates of Monocyte Activation and HIV persistence in Methamphetamine Users. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, 14(1), 16-22.
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6

Detecting EGFR Mutations in Cellular DNA

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Cellular DNA was examined for EGFR mutations within exons 18 through 21 using the peptide nucleic acid–locked nucleic acid PCR clamp method.12 (link) The exon‐19 mutation encoding the D761Y mutation was detected by DNA sequencing, as described previously.13 (link) The C797S mutation was detected by performing digital droplet PCR (ddPCR) using a Bio‐Rad QX200 ddPCR instrument, as previously described.11 (link)
Fukuda K., Otani S., Takeuchi S., Arai S., Nanjo S., Tanimoto A., Nishiyama A., Naoki K, & Yano S. (2021). Trametinib overcomes KRAS‐G12V–induced osimertinib resistance in a leptomeningeal carcinomatosis model of EGFR‐mutant lung cancer. Cancer Science, 112(9), 3784-3795.
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