Quantstudio 3d digital pcr system

Digital PCR was performed by using a QuantStudio™ 3D digital PCR system (Life Technologies, Carlsbad, CA) to detect human TET1, TET2, TET3, DNMT1, DNMT3a, DNMT3b, and GAPDH according to the manufacturer's instructions. We used TaqMan^® Assay ID probe Hs00286756_m1, Hs00325999_m1, Hs00379125_m1, Hs00154749_m1, Hs01027166_m1, Hs00171876_m1, Hs99999905_m1, respectively. For Digital PCR, 80 ng for TET1, TET2, TET3, DNMT1, DNMT3a and DNMT3b or 1 ng for GAPDH of cDNA were applied to following protocol: 96 °C for 10 min, 39 cycles at 60 °C for 2 min, 98 °C for 30 s, and 60 °C for 2 min. The data was analyzed with the QuantStudio™ 3D AnalysisSuite™ v3.0 for quantification of each gene's copy numbers.

Standard DNA templates were generated from either PCR products or genomic DNA. To construct an amplicon-based DNA standard, DNA fragments (594 bp at the locus from 54772 to 55365 in Acc. No. AL161450) containing the wild-type JAK2 or V617F mutant were first PCR-amplified from genomic DNA using the FO and RO primers. The concentrations of the purified PCR products were measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific). The amplicon-based DNA standard was then prepared by mixing each amplicon to obtain JAK2V617F allele frequencies of 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, and 10%. To construct the genomic DNA standard, genomic DNA was extracted from JAK2 wild-type UT-7/EPO and JAK2V617F homozygous HEL cells [14 (link), 15 (link)] using a QIAamp DNA Mini kit (Qiagen) according to the manufacturer’s instructions. The concentration and JAK2 copy number of each extracted DNA sample were measured using a Quantus Fluorometer (Promega) and the QuantStudio 3D digital PCR system (Life Technologies), respectively. Genomic DNA standards with JAK2V617F allele frequencies of 0%, 0.01%, 0.05%, 0.5%, and 1% were prepared by mixing the proper amounts of each type of genomic DNA. All of the genomic DNA standards were prepared at the following concentrations: 10, 50, and 100 ng/μL.

The chip-based digital PCR (dPCR) was conducted on a QuantStudio 3D Digital PCR System (Life Technologies) for the absolute quantification of the copy number of circVPS13A and miR-182. cDNA was synthesized from RNA isolated from EGC under indicated conditions, and digital PCR was conducted using the following reaction conditions: hot start at 96°C for 10 min, denaturation at 98°C for 30 s, annealing/extension at 62°C for 2 min for a total of 39 cycles, followed by a final extension step at 65°C for 2 min. The data analysis was performed with QuantStudio 3D AnalysisSuite Cloud Software version 3.1.2. The primers used are:
circVPS13A F, 5′-TGAAATTCTTGCAGAAATGTTG-3′, R, 5′-GTGCTCCTGGTCTTTGCACAAT-3′;
miR-182 F, 5′-ATCACTTTTGGCAATGGTAGAACT-3′, R, 5′-TATGGTTTTGACGACTGTGTGAT-3′.

Digital PCR was used to confirm the presence of somatic mutations (GNAQ p.Arg183Gln; GNAQ p.Arg183Gly) in the affected tissue samples. DNA from matched control tissues and one negative healthy control sample from peripheral blood were also evaluated. A custom TaqMan SNP Genotyping Assay was optimized for each variant using the QuantStudio 3D Digital PCR System (Life Technologies, Grand Island, NY). Results were analyzed using the QuantStudio 3D AnalysisSuite software relative quantification module.

A QuantStudio 3D Digital PCR system (Life Technologies, Carlsbad, CA, USA) was used to estimate the population of cells expressing specific markers for each cell type expected in culture. Every chip was loaded with 2 Taqman assay primers: one with FAM signal for the specific targeted gene and one with VIC signal for the housekeeping gene POLR2A. The chip load was completed following the manufacturer’s manual with commercially available dPCR Master Mix and cDNA from RNA extracted from Snap Frozen cells. The quantity of cDNA loaded in each 20,000 wells chip was 50 ng. Assuming each mammalian cells has around 10–30 pg of total RNA, each well should represent at most one cell with a significant number of empty wells as a safety margin. Cycling conditions were 95 °C for 10 min, then 40 cycles of 95 °C for 15 s, and 60 °C for 1 min.

We validated mutations of 12 genes either by digital-PCR or Sanger sequencing as described previously [50 (link)]. Digital-PCR was performed using the TaqMan Genotyping assay and the QuantStudio 3D digital PCR system (Life Technologies) according to the manufacturer's protocol.

Genomic DNA was extracted from PBMCs 24 hours after infection with NL4.3-Renilla, using DNeasy Blood and Tissue kit (Qiagen), and quantified using the Nanodrop-1000 spectrophotometer (Nanodrop). The samples were measured by the QuantStudio 3D Digital PCR System (Life Technologies). The reaction mixture for Digital PCR (dPCR) is as follows: cDNA, QuantStudio 3D Digital PCR Master Mix v2, 300nM of C1_2LTR primer, 300nM C4R_71 primer, 250nM 2nr4nr_FAM probe, 0,5x CCR5_VIC probe and H₂O for a final volume of 14.5 μl. The digital PCR reaction mix was loaded onto the QuantStudio 3D digital PCR chips, according to the manufacturer instructions. The thermal cycling and amplifications were performed in a ProFlex 2xFlat PCR system, according to the manufacturer protocol: 96°C for 10 min, followed 39 cycles of 55 °C for 2 min and 98°C for 30 sec, 55 °C for 2 min and a stabilization phase at 22°C. The chips were transferred to a QuantStudio 3D digital PCR instrument for imaging and the final analysis of the files generated was carried out using the cloud-based.