Each DNA sample was diluted to 1000 ng/mL, as measured by a Qubit 2.0 Fluorometer (Thermo Fisher Scientific). PCR reaction mixtures contained 9 μL QuantStudio 3D Digital PCR Master Mix (Thermo Fisher Scientific), 0.45 μL TaqMan assay, and 8.55 μL diluted DNA. Fifteen μL of the 18 μL reaction mixture was loaded in a QuantStudio 3D Digital PCR 20K chip (Thermo Fisher Scientific) and amplified using the GeneAmp PCR system 9700 (Thermo Fisher Scientific) as follows: 96°C for 10 minutes, then 39 cycles of 56°C for 2 minutes, 98°C for 30 seconds, and a final extension step at 60°C for 2 minutes. Commercial primers (wet lab‐validated Custom TaqMan SNP Genotyping Assays) were used.
Quantstudio 3d digital pcr 20k chip
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio 3D Digital PCR 20K Chip is a microfluidic chip designed for use with the QuantStudio 3D Digital PCR System. The chip has a capacity of 20,000 reaction wells and is used for digital PCR (dPCR) analysis.
Automatically generated - may contain errors
Lab products found in correlation
Quantstudio 3d digital pcr master mix, by Thermo Fisher Scientific (3 mentions)
Quantstudio 3d analysissuite cloud software, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3d digital pcr master mix v2, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d chip reader, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d analysissuite software, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Quantstudio 3d digital pcr instrument, by Thermo Fisher Scientific (1 mentions)
Proflex 2x flat pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman pri mirna assay, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d digital pcr chip loader, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d digital pcr system platform, by Thermo Fisher Scientific (1 mentions)
8 protocols using quantstudio 3d digital pcr 20k chip
1
Digital PCR Quantification Protocol
2
Quantitative Digital PCR for Dystrophin
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cDNA samples were diluted 1:25 yielding a final concentration of 2 ng/ul. Next, 15μl reactions using QuantStudio 3D Digital PCR Master Mix v2 (A26358, ThermoFisher) were loaded onto Quantstudio 3D digital PCR 20K Chip (A26316, ThermoFisher) using the Quantstudio chip loading device. Primer probes for VIC-labeled Dmd exon 2–3 and FAM-labeled skipped Dmd exon 22–24 were diluted 20-fold (1x, 5% reaction volume). For ddPCR we used the following cycling conditions: 96°C for 10 minutes, followed by 39 cycles of denaturation at 98°C for 30 seconds and annealing/extension at 60°C for 2 minutes. Cycled chips were loaded into Quantstudio 3D chip reader and resulting data was imported into QuantStudio 3D Analysis Suite Cloud Software (ThermoFisher). For analysis, we generated a copy number for each labeled probe in our samples. Data corresponds to absolute copy number of skipped Dmd transcript per ng RNA.
3
Quantitative Digital PCR Assay Protocol
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Comparative genomic hybridizations assay was adapted as described by McHale et al. (2012 ) and Dobbels et al. (2017 ). The Taqman assay was performed according to Kadam et al. (2016 ). The digital PCR was performed according to Wan et al. (2016 ). Briefly, 20 μL reaction was prepared, consisting of 10 μL 2× master reaction mix (Life Technologies, Waltham, MA), 1 μL assay mix (18 μm Forward and 18 μm reverse primers + 5 μm probe), 1 μL DNA (final concentration 40 ng) and 9 μL ddH2O. The forward and reverse probe/primer sequences were used according to Kadam et al. (2016 ). A 14.5 μL of the PCR mixture was loaded onto a QuantStudio™ 3D Digital PCR 20K Chip (Thermo Fisher Scientific, Waltham, MA). The chip was covered with immersion fluid, a lid was applied, the assembly was filled with immersion fluid and the loading port was sealed according to the manufacturer's instructions. The chips were loaded into the Dual Flat Block GeneAmpR PCR System 9700 (Life Technologies), and PCR was performed using the following conditions: 96 °C for 10 min; 60 °C for 2 min and 98 °C for 30 s, for 39 cycles; 60 °C for 2 min; 10 °C for storage. The Digital PCR 20K Chip was read using the QuantStudio™ 3D Digital PCR Chip Reader, and the data was analysed using the QuantStudio™ 3D AnalysisSuite™ Software (Thermo Fisher Scientific, Waltham, MA).
4
Quantitative Digital PCR for KRAS Mutation Detection
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dPCR combined with asymmetric PCR was performed using the molecular beacons in the wells. The sequences of the employed primers and molecular beacons are described in Table 1 . Samples for dPCR were prepared by mixing 1x QuantStudio 3D Digital PCR Master Mix v2 (Thermo Fisher Scientific), 0.25 μM forward primer, 0.5 μM reverse primer, 0.5 μM each of molecular beacons for the WT, G12R-mutant and G12D-mutant KRAS, and DNA template containing approximately 1 × 103 copies of the KRAS gene in a final reaction volume of 15 μl. The samples were divided into 20,000 wells on a QuantStudio 3D Digital PCR 20 K Chip (Thermo Fisher Scientific). Amplification in the wells was carried out by the thermal cycler as follows: 10 min at 95 °C and 60 cycles of 15 s at 95 °C and 75 s at 60 °C.
5
Quantitative Analysis of AR Isoforms
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For each RNA sample, 12 µL of the template were retrotranscribed to cDNA using a QuantiTect Reverse Transcription Kit (Qiagen, Venlo, The Netherlands) for a final volume of 20 µL. The assessment of AR-V7, AR-FL and RNY4 copies was performed on cDNA using custom assays (primers described elsewhere) [12 (link),14 (link)]. DNA was employed to detect the AR T878A variant (TaqMan™ SNP Genotyping Assay C_175239649_10, Thermo Fisher Scientific, MA, USA), AR gene copy numbers with RNaseP as the internal reference standard (TaqMan™ Copy Number Assay Hs04107225_cn and TaqMan™ Copy Number Reference Assay, human, RNase P, Thermo Fisher Scientific, USA). A volume of 15 μL of the reaction mix was loaded on a QuantStudio 3D Digital PCR 20K Chip (Thermo Fisher Scientific, MA, USA) and thermocycling ran at 95 °C for 8 min, 40 cycles (37 cycles for the AR-V7 assay) at 95 °C for 15 s and at 60 °C for 1 min, with a final extension step at 60 °C for 2 min. Copies/µL reaction were retrieved with QuantStudio 3D AnalysisSuite Cloud Software and copies/mL plasma were calculated by multiplying this value by the dilution factors applied during the process.
6
Digital PCR Analysis of Alcohol-Induced miRNA Expression
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30 ng/μL of each alcohol exposure sample was tested with three TaqMan Pri-miRNA assays (Mm04227702 pri-mmu-mir-9-1, Mm03306269 pri-mmu-mir-9-2, and Mm03307250 pri-mmu-mir-9-3) (Thermo Fisher Scientific Inc.). 1μL of each sample was added to 10 μL QuantStudio 3D Digital PCR Master Mix, 1 μL of TaqMan Assay (20X), and 8 μL of nuclease-free water for 20 μL of the reaction mix. 14.5 μL of reaction mix was loaded on each QuantStudio 3D Digital PCR 20K Chip (Thermo Fisher Scientific Inc.) using QuantStudio 3D Digital PCR Chip Loader (Thermo Fisher Scientific Inc.) according to manufacturer’s instruction. The digital PCR was performed on Proflex 2x Flat PCR System (Thermo Fisher Scientific Inc.) with thermal cycling of 10 min at 96°C, followed by 39 cycles at 60°C for 2 min and 98°C for 30 s, followed by holding at 60°C for 2 min and 10°C for long term. Each chip fluorescence intensity was read using QuantStudio 3D Digital PCR instrument (Thermo Fisher Scientific Inc.) and analyzed copies/μL based on Poisson distribution using QuantStudio 3D Analysis Suite Cloud Software (Thermo Fisher Scientific Inc.).
7
Digital PCR Protocol for BRAF and KRAS Mutation Detection
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Digital PCR experiment set up was performed according to the Digital MIQE Guidelines37 .
Amplification was carried out in 15 µl volume using the QuantStudio 3D Digital PCR System Platform (Thermo Fisher Scientific, Carlsbad, CA, USA). PCR reaction mix contained 8 µl of 2X QuantStudio 3D Digital PCR Master Mix (Thermo Fisher Scientific), 0.4 µl of 40X TaqMan-MGB-FAM-probe assay (BRAF assay ID: BRAF_476_mu; KRAS G12S Assay ID: Hs000000048; KRAS G13D assay ID: Hs000000056_rm; Thermo Fisher) 1.1 µl of diluted DNA (50 ng/µl) and 6.5 µl of nuclease-free water. Serial dilutions of genomic DNA standards (Cat Num. HD238, Horizon Discovery) and of negative controls with DNA-free water were used to estimate the limit of detection. (Suppl. Fig.3 ).
To amplify the target genes, 15 µl of the reaction mix were loaded onto a QuantStudio 3D Digital PCR 20 K Chip (Thermo Fisher Scientific) and run following this PCR program: 95 °C for 8′, 40 cycles at 95 °C for 15″ and 60 °C for 1′, followed by a final extension step at 60 °C for 2′. Raw data were analyzed with QuantStudio 3D Analysis Suite Cloud Software to calculate gene copies per ml.
Amplification was carried out in 15 µl volume using the QuantStudio 3D Digital PCR System Platform (Thermo Fisher Scientific, Carlsbad, CA, USA). PCR reaction mix contained 8 µl of 2X QuantStudio 3D Digital PCR Master Mix (Thermo Fisher Scientific), 0.4 µl of 40X TaqMan-MGB-FAM-probe assay (BRAF assay ID: BRAF_476_mu; KRAS G12S Assay ID: Hs000000048; KRAS G13D assay ID: Hs000000056_rm; Thermo Fisher) 1.1 µl of diluted DNA (50 ng/µl) and 6.5 µl of nuclease-free water. Serial dilutions of genomic DNA standards (Cat Num. HD238, Horizon Discovery) and of negative controls with DNA-free water were used to estimate the limit of detection. (Suppl. Fig.
To amplify the target genes, 15 µl of the reaction mix were loaded onto a QuantStudio 3D Digital PCR 20 K Chip (Thermo Fisher Scientific) and run following this PCR program: 95 °C for 8′, 40 cycles at 95 °C for 15″ and 60 °C for 1′, followed by a final extension step at 60 °C for 2′. Raw data were analyzed with QuantStudio 3D Analysis Suite Cloud Software to calculate gene copies per ml.
8
Absolute Quantification of BCR-ABL1 Transcripts by dPCR
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BCR-ABL1 transcript absolute quantification was performed by digital PCR (dPCR), due to its reported sensitivity in human BCR-ABL1 transcript detection [50 (link)]. dPCR assay was performed by the chip-based dPCR platform system Quant Studio 3D (QS3D) (Thermo Fisher Scientific), as previously described [51 (link),52 (link),53 (link)]. Briefly, a reaction mix containing 8 μL of 2X QuantStudio 3D Digital PCR Master Mix (Thermofisher Scientific), 0.8 μL of 20X TaqMan-MGB-FAM-probe customized assay, 5 μL of cDNA, and 2.2 μL of nuclease-free water was prepared. The negative control reaction mix contained 8 µL of 2X QuantStudio 3D Digital PCR Master Mix, 0.8 μL of 20X TaqMan-MGB-FAM-probe assay, and of 7.2 μL nuclease-free water. For each sample, we loaded 15 μL of the reaction mix onto a QuantStudio 3D Digital PCR 20K Chip (Thermofisher Scientific) by the automatic chip loader. The target was amplified using the following thermocycling profile: 95 °C for 8′, 45 cycles at 95 °C for 15”, and 60 °C for 1′, with a final extension step at 60 °C for 2′ on ProFlex PCR system. The chips were then imaged by dPCR QS3D and a secondary analysis was performed by QuantStudio 3D AnalysisSuite Cloud Software. Positivity emission threshold was fixed at 4000 RFU, as previously reported [54 (link)].
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