Qx100 droplet digital pcr ddpcr system

Manufactured by Bio-Rad

Sourced in United States

The QX100 droplet digital PCR (ddPCR) system is a laboratory instrument designed for precise and sensitive nucleic acid quantification. It partitions samples into thousands of nanoliter-sized droplets, allowing for the detection and quantification of target DNA or RNA molecules with high precision. The QX100 system provides a robust and reproducible digital PCR platform for applications such as gene expression analysis, rare mutation detection, and copy number variation studies.

Automatically generated - may contain errors

Lab products found in correlation

9 protocols using qx100 droplet digital pcr ddpcr system

Quantification of Plasmid DNA Standards by ddPCR

Check if the same lab product or an alternative is used in the 5 most similar protocols

For quantification of the plasmid standards, a QX100 droplet digital PCR (ddPCR) system was applied (Bio-Rad, Hercules, USA). A total of 2 μL of each plasmid DNA (1:10⁶ diluted) were added to 18 μL of ddPCR reaction mix containing 1x ddPCR supermix (Bio-Rad, Hercules, USA) and primers and probes dissolved in PCR grade water (final concentrations see Table 2). Water served as non-template control. Droplets were generated using 8-well cartridges in a droplet generator (Bio-Rad, Hercules, USA) and then transferred to a 96-well plate using a multichannel pipette. End-point PCR was performed using a T100 thermal cycler (Bio-Rad, Hercules, USA) under the following conditions: 10 min initial denaturation at 95 °C, 45 cycles of 94 °C for 30 s and 60 °C for 1 min, and finally 10 min at 98 °C A heating ramp rate of 2 °C per second was applied. After amplification, droplet separation, counting and fluorescence measurement were performed in the QX100 Droplet Reader (Bio-Rad, Hercules, USA). The QuantaSoft software (Bio-Rad, Hercules, USA; version 1.7.4) was used for data acquisition and analysis.

Guertler P., Grohmann L., Naumann H., Pavlovic M, & Busch U. (2019). Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179. Biomolecular Detection and Quantification, 17, 100076.

+ Open protocol

+ Expand

Droplet digital PCR for ESR1 mutations

Check if the same lab product or an alternative is used in the 5 most similar protocols

Bio-Rad QX100 Droplet Digital PCR (ddPCR) System was used for testing mutation abundance. Validation of the sequencing data was performed with primers and probes designed for D538G and wild type (WT). WT probe: CCCCTCTATGaCCTGCT-HEX, D538G mutant: CTCTATGgCCTGCTGC-FAM 900 nM for ESR1 F primer: TACAGCATGAAGTGCAAG, and ESR1 R primer: TGGGCGTCCAGCA were also used. The local recurrence cohort was examined using Bio-Rad-specific kits for D538G, L536R, and Y537S/N/C mutations. Digital PCR conditions were optimized with a temperature gradient to identify the optimal annealing/extension temperature on a QX200 ddPCR system (Bio-Rad) using TaqMan chemistry. Ten to 50 ng DNA from FFPE samples were used in each reaction, with the addition of 10 μl of ddPCR Supermix for probes (no dUTP) (Bio-Rad) and 250 nM TaqMan WT probe and 250 nM TaqMan D538G probe in a volume of 20 μl for each reaction. The emulsified PCR reaction was partitioned into ~ 14,000 droplets per sample in a QX100 droplet generator according to the manufacturer’s instructions.

Zundelevich A., Dadiani M., Kahana-Edwin S., Itay A., Sella T., Gadot M., Cesarkas K., Farage-Barhom S., Saar E.G., Eyal E., Kol N., Pavlovski A., Balint-Lahat N., Dick-Necula D., Barshack I., Kaufman B, & Gal-Yam E.N. (2020). ESR1 mutations are frequent in newly diagnosed metastatic and loco-regional recurrence of endocrine-treated breast cancer and carry worse prognosis. Breast Cancer Research : BCR, 22, 16.

+ Open protocol

+ Expand

Quantifying Expression of WRKY33, PDF1.2, VQ16

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Quantification of expression of WRKY33, PDF1.2 and VQ16 were conducted by Droplet Digital PCR (ddPCR) according to the methods described previously^{66 (link)}. RNA of Lm infected and mock inoculated Topas and Westar (from Group I), W-LepR1 (from Group II), T-LepR2 (from Group III) and T-LepR1 (from Group VI) at 3, 6, 9 dai were adjusted to 1 μg of RNA, and cDNA from three biological replicates was synthesized using an iScript™ Advanced cDNA Synthesis Kit according to the manufacturer’s protocol (Bio‐RAD). All primers and probes (Supp. Table 2) were designed using “Quest tool” complimented by IDT (https://www.idtdna.com/PrimerQuest/Home/Index) and “BnActin” was used as reference. ddPCR was performed using a QX100 Droplet Digital PCR (ddPCR™) System – Bio-Rad. The Bio-Rad QuantaSoft™ Analysis Pro (QuantaSoft AP) software was used to calculate the ratio signal of assay/Actin. In order to get RNAseq and ddPCR result comparable, heat map was generated based on: Log2 (Assay/Actin) (mean of replicates): inoculated with Lm/not inoculated control and Log2 RPKM (mean of replicates): inoculated with Lm/not inoculated control (Supp. Fig. 7).

Haddadi P., Larkan N.J, & Borhan M.H. (2019). Dissecting R gene and host genetic background effect on the Brassica napus defense response to Leptosphaeria maculans. Scientific Reports, 9, 6947.

+ Open protocol

+ Expand

Detecting CCNE1 Amplification and RB1 Mutation

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Genomic DNA was extracted from cells and formalin-fixed paraffin-embedded samples with the DNeasy Blood and Tissue Kit (Qiagen) as per the manufacturer’s instructions. The detection of cylcin E1 amplification by digital PCR was performed with a CCNE1 Taqman Copy Number Variation Assay (Hs07158517_cn) and a TERT TaqMan Copy Number Reference Assay (4403316) from Life Technologies on a QX-100 droplet digital PCR (ddPCR) system (Bio-Rad). To detect RB1 pM695fs^*26, we designed a primer probe combination targeting RB1 c.2083-2084insA: pM695fs^*26. Digital PCR was performed as described previously (18 , 19 (link)). The ratio of CCNE1:TERT was calculated using the Poisson distribution in QuantaSoft. The RB1 pM695fs^*26 fraction was assessed as published previously (18 ).

Herrera-Abreu M.T., Palafox M., Asghar U., Rivas M.A., Cutts R.J., Garcia-Murillas I., Pearson A., Guzman M., Rodriguez O., Grueso J., Bellet M., Cortés J., Elliott R., Pancholi S., Lord C.J., Baselga J., Dowsett M., Martin L.A., Turner N.C, & Serra V. (2016). Early Adaptation and Acquired Resistance to CDK4/6 Inhibition in Estrogen Receptor–Positive Breast Cancer. Cancer research, 76(8), 2301-2313.

+ Open protocol

+ Expand

Quantification of Circulating Donor cfDNA

Check if the same lab product or an alternative is used in the 5 most similar protocols

cfDNA was isolated from 3 ml of anti-coagulated blood plasma by using the Circulating Nucleic Acid kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The QX100 droplet digital PCR (ddPCR) system (Bio-Rad Laboratories, CA, United States) was used for the quantification of (dd)cfDNA. Samples of 20 μl were prepared for PCR reactions by making a mixture containing purified cfDNA, water, a donor specific target assay (discriminative SNP) and ddPCR Supermix for Probes (Bio-Rad). Droplets were generated with a QX100 droplet generator (Bio-Rad) according to the manufacturer’s instructions. The ddPCR was performed using the T100^TM Thermal Cycler (Bio-Rad) with the following amplification protocol: 95°C for 10 min, 40× (94° for 30 s, 55° for 1 min), then 98°C for 10 min. The quantified droplets were analyzed through a QX100 droplet reader (Bio-Rad) using Quantasoft software version 1.0.596 (Bio-Rad). ddcfDNA values were quantified either as fraction (%) (donor-specific SNP signal/total SNP signal (donor-specific SNP signal + non-donor-specific SNP signal)) or as concentration (copies/ml plasma). In samples were ddcfDNA was quantified with two or three SNPs, the ddcfDNA values were averaged.

Verhoeven J.G., Hesselink D.A., Peeters A.M., de Jonge E., von der Thüsen J.H., van Schaik R.H., Matic M., Baan C.C., Manintveld O.C, & Boer K. (2022). Donor-Derived Cell-Free DNA for the Detection of Heart Allograft Injury: The Impact of the Timing of the Liquid Biopsy. Transplant International, 35, 10122.

+ Open protocol

+ Expand

Droplet Digital PCR for lncRNA and mRNA Validation

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Validation of selected lncRNA and mRNA transcripts was performed on 12 pairs of pre-ischemic and post-ischemic samples using the Bio-Rad QX100™ Droplet Digital PCR (ddPCR™) system (Pleasanton, CA).^{24 (link)} cDNA was generated from patient samples as described above. PCR reactions were constructed using custom probe and primer sequences (Table S1) and absolute concentration of transcripts was obtained according to the manufacturer’s specifications. Relative expression was calculated using the house keeping gene tumor protein, translationally-controlled 1 (TPT1). Statistical significance was determined using a paired t-test. Stripcharts and statistics were generated using the R package ggplots2.²⁵

Saddic L.A., Sigurdsson M.I., Chang T.W., Mazaika E., Heydarpour M., Shernan S.K., Seidman C.E., Seidman J.G., Aranki S.F., Body S.C, & Muehlschlegel J.D. (2017). The Long Noncoding RNA Landscape of the Ischemic Human Left Ventricle. Circulation. Cardiovascular genetics, 10(1), e001534.

+ Open protocol

+ Expand

Droplet Digital PCR for KRAS Mutation Detection

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total genomic DNA (gDNA) was extracted directly from the sample material on the slide used for enumeration (QIAamp DNA Micro Kit, Qiagen). The QX100 Droplet Digital PCR System (ddPCR, Biorad), PrimePCR KRAS mutant, and WT assays (Biorad, dHsaCP2000001 (G12D), dHsaCP2000002 (G12D WT), dHsaCP2000005 (G12V), dHsaCP2000006 (G12V WT), dHsaCP2000009 (G12R), and dHsaCP2000010 (G12R WT)) were used to detect the following KRAS mutations in gDNA: G12D, G12V, and G12R. A total 50 ng of gDNA was used for each PCR. PDAC 215 (G12D), PDAC 247 (G12V), and PDAC JH033 (G12R) were used for positive controls and leukocytes of a healthy donor served as a negative control. In ddPCR, the samples containing gDNA were partitioned into 20,000 droplets and loaded into thermo cycler. Following PCR amplification, droplets from each sample are streamed in single file through the droplet reader. Absolute concentration of KRAS mutant and WT DNA copies were determined using the QuantaSoft software provided by the manufacturer. Briefly, positive droplets which contain at least one copy of the target exhibit increased fluorescence. The system detects Mutant (HEX) and WT (FAM) alleles by counting the number of droplets positive for each fluorophore.

Agerbæk M.Ø., Bang-Christensen S.R., Yang M.H., Clausen T.M., Pereira M.A., Sharma S., Ditlev S.B., Nielsen M.A., Choudhary S., Gustavsson T., Sorensen P.H., Meyer T., Propper D., Shamash J., Theander T.G., Aicher A., Daugaard M., Heeschen C, & Salanti A. (2018). The VAR2CSA malaria protein efficiently retrieves circulating tumor cells in an EpCAM-independent manner. Nature Communications, 9, 3279.

+ Open protocol

+ Expand

Quantifying Total HIV-1 DNA

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

PBMCs were subjected to DNA extraction using commercial kits (QIAGEN AllPrep DNA/RNA Mini Kit or QIAGEN DNeasy kit; Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions. Total HIV-1 DNA was amplified using the QX100 Droplet Digital PCR System (ddPCR; Bio-Rad, Hercules, California, USA) using primers and probes described previously [11 (link)].

Moraka N.O., Garcia-Broncano P., Hu Z., Ajibola G., Bareng O.T., Pretorius-Holme M., Maswabi K., Maphorisa C., Mohammed T., Gaseitsiwe S., VanZyl G.U., Kuritzkes D.R., Lichterfeld M., Moyo S, & Shapiro R.L. (2021). Patterns of pretreatment drug resistance mutations of very early diagnosed and treated infants in Botswana. AIDS (London, England), 35(15), 2413-2421.

+ Open protocol

+ Expand

Quantifying HIV-1 DNA in PBMCs

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

PBMCs were subjected to DNA extraction using commercial kits (QIAGEN AllPrep DNA/RNA Mini Kit or QIAGEN DNeasy kit) according to the manufacturer’s instructions. Total HIV-1 DNA was amplified using the QX100 Droplet Digital PCR System (ddPCR; Bio-Rad) using primers and probes described previously (65 (link)) [127–base pair (bp) 5′-LTR–gag amplicon; coordinates 684 to 810 in HIV-1 reference strain HXB2) and normalized to the RPP30 gene. PCR was performed using the following program: 95°C for 10 min, 45 cycles of 94°C for 30 s and 60°C for 1 min, 72°C for 1 min. Data were analyzed using the QuantaSoft software (Bio-Rad). When viral copies where undetectable, data were reported as “limit of detection” (LOD), calculated as 0.2 copies per maximum number of cells tested without target identification.

Garcia-Broncano P., Maddali S., Einkauf K.B., Jiang C., Gao C., Chevalier J., Chowdhury F.Z., Maswabi K., Ajibola G., Moyo S., Mohammed T., Ncube T., Makhema J., Jean-Philippe P., Yu X.G., Powis K.M., Lockman S., Kuritzkes D.R., Shapiro R, & Lichterfeld M. (2019). Early antiretroviral therapy in neonates with HIV-1 infection restricts viral reservoir size and induces a distinct innate immune profile. Science translational medicine, 11(520), eaax7350.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!