Two nanograms of ctDNA or buffy coat DNA was subjected to targeted high-fidelity preamplification for 15 cycles using custom-designed primers (Additional file 3 : Table S2) and PCR conditions previously described [13 (link)]. Targeted preamplification products were purified using QIAquick PCR Purification kit (Qiagen) and diluted at 1:20 before use in ddPCR reaction. 1.5 μl of diluted preamplified DNA was used as input for ddPCR reaction. ddPCR was performed for ESR1-D538G, FOXA1-Y175C, and PIK3CA-H1047R mutations. Custom ddPCR assays were developed for ESR1-D538G (Integrated DNA Technologies) and FOXA1-Y175C (ThermoFisher Scientific). Sequences are described in Additional file 4 : Table S3. PIK3CA-H1047R was analyzed using PrimePCR ddPCR assay (Bio-Rad Laboratories) dHsaCP2000078 (PIK3CA)/dHsaCP2000077 (H1047R). Nuclease-free water and buffy coat-derived wildtype germline DNA as negative controls, and oligonucleotides carrying mutation of interest or DNA from a cell line with mutation as positive controls, were included in each run to eliminate potential false-positive mutant signals. An allele frequency of 0.1% was used as a lower limit of detection.
Primepcr ddpcr assay
Manufactured by Bio-Rad
Sourced in Israel, United States
PrimePCR ddPCR assays are a set of pre-designed, gene-specific primers and probes for use with droplet digital PCR (ddPCR) systems. They enable precise and sensitive quantification of target DNA or RNA sequences in a variety of sample types.
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5 protocols using primepcr ddpcr assay
1
Targeted Preamplification and ddPCR for ctDNA Analysis
2
Detecting ADNP Gene Mutations using ddPCR
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20 μl ddPCR reactions were prepared (Bio-Rad Laboratories Ltd. Rishon Le Zion, Israel & Prague Czech Republic): including 10 μl of ddPCR™ Supermix for Probes (No dUTP), 1 μl of PrimePCR™ ddPCR™ Assay, 120 ng DNA, and nuclease-free water. Two targets were detected simultaneously with different fluorescence probes: the FAM probe detected the mutated sequence NM_001282531.2(ADNP):c.2188C>T (p.Arg730*), (unique assay Id: dHsaMDS971559989), or in separate reactions, the NM_015339.3(ADNP):c.2157C>G (p.Tyr719*) (unique assay Id: dHsaMDS423261257, supplemental methods). The HEX probe was used to detect the control sequence (Gene Id: 23394). The annealing temperature for various sets of primers and probes was established by previous gradient runs. After droplet generation, PCR was performed in a C1000 as follows, 95 °C 10 min, 40 cycles of 94 °C 30 s, 54 °C 1 min, 98 °C 10 min. The droplets were analyzed with a QX200 instrument, and the data were processed by the Quantasoft software, with the rare event detection (RED) mode according to the digital MIQE guidelines [32 (link)].
3
Droplet Digital PCR for KRAS Mutation
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KRAS mutations were analyzed on DNA from the tumor samples as well as on plasma DNA. The ddPCR method was previously reported [31 (link)]. The analysis was performed as described above for meth-HOXA9, except for the bisulfite conversion of the DNA. We used PrimePCR ddPCR assays (Bio-Rad, Hercules, CA, USA) for the specific mutations in the tumor tissue harbored by the patient, as detected by NGS.
4
Droplet Digital PCR for KRAS Mutations
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Genotypes with G12D, G12S, G13C, and G13D were analyzed by droplet digital PCR (ddPCR) in discordant cases of KRAS. The following PrimePCR ddPCR assays (BioRad Laboratories, Inc., Hercules, CA) were analyzed: KRAS p.G12D (dHsaCP2500596) and KRAS WT for p.G12D (dHsaCP2500597); KRAS p.G12S (dHsaCP2500588) and KRAS WT for p.G12S (dHsaCP2500589); KRAS p.G13C (dHsaCP2500594) and KRAS WT for p.G13C (dHsaCP2500595); and KRAS p.G13D (dHsaCP2500598) and KRAS WT for p.G13D (dHsaCP2500599). For each ddPCR reaction, 20 ng of DNA was used. Each reaction included a positive control (KRAS p.G12D) using the HD780 Reference Standard Set (Horizon, Cambridge, UK). All experiments were performed according to the manufacturer’s instructions, as previously described [10 (link)]. Samples that contained at least two droplets in the 6-fluorescein amidite–positive area were considered positive.
5
Droplet Digital PCR for Rare Mutations
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For all ddPCR runs, matched cfDNA and genomic DNA (g-DNA) derived from buffy coat were included to confirm that the mutation was somatic. Purified WG-RCA DNA was heated at 95 °C for 5 min and then cooled on ice prior to analysis. Approximately 75 ng of WG-RCA DNA, 7.5–75 ng of buffy gDNA, and 1–14 ng of unamplified cfDNA were used per ddPCR reaction. For buffy gDNA and WG-RCA DNA, restriction enzyme was included during droplet generation using the QX100 Droplet Digital PCR System (Bio-Rad Laboratories). No template control and g-Blocks bearing mutation of interest or DNA from a cell line with mutation as positive controls were included in each run. Allele frequency of 0.1% was used as a lower limit of detection based on background mutant signal detected in some buffy gDNA samples. The following PrimePCR ddPCR assays (Bio-Rad Laboratories) were analyzed: dHsaCP2000078 (PIK3CA)/dHsaCP2000077 (H1047R), dHsaCP2000076 (PIK3CA)/dHsaCP2000075(E545K), and dHsaCP2000002 (KRAS)/dHsaCP2000001(G12D). Custom ddPCR assays were developed for PIK3CA-N345K (Life Technologies), ESR1-D538G (Integrated DNA Technologies), and ESR1-Y537C (Life Technologies) as described in Supplementary Table S5 .
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