Digital PCR (dPCR) was carried out by mixing 3.33 μl diluted template DNA (50 ng/μl, diluted 1:10 for E11 and E18, 1:2 for PN1 and undiluted for 3 M hippocampus); concentrations of DNA are described below and varied by experiment with 16.5 μl QuantStudio 3D master mix, 3.33 μl 20× gene expression assay, and 9 μl water [32.16 μl (enough for two chips with excess)] (Life Technologies). Reactions were loaded onto QuantStudio 3D digital PCR chips (20,000 wells per chip) using the QuantStudio 3D chip loader according to manufacturer’s instructions (Life Technologies). Chips were then sealed and cycled on a GeneAmp PCR system 9700 with a flatblock attachment using the following conditions: Stage 1—96 °C for 10 min, Stage 2—60 °C for 2 min and then 98 °C for 30 s (repeat Stage 2 39 times), Stage 3—60 °C for 2 min and an infinite 10 °C hold. Chips were then read in the QuantStudio 3D chip reader (Life Technologies) to obtain raw fluorescent values per well. Quality check of the chips and counting of positive and negative wells in order to determine copies/microliter were carried out on the QuantStudio 3D AnalysisSuite cloud software (LifeTechnologies) using the absolute quantification module and then calculated to copies per μg of input RNA including incorporating dilutions stated above.
Quantstudio 3d digital pcr chip
Manufactured by Thermo Fisher Scientific
The QuantStudio 3D Digital PCR Chip is a lab equipment product designed for digital PCR analysis. It provides a platform for precise quantification of nucleic acid targets. The chip enables partitioning of samples into thousands of individual reaction chambers for parallel amplification and detection of target sequences.
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Lab products found in correlation
Quantstudio 3d digital pcr master mix, by Thermo Fisher Scientific (4 mentions)
Quantstudio 3d digital pcr instrument, by Thermo Fisher Scientific (4 mentions)
Quantstudio 3d digital pcr system, by Thermo Fisher Scientific (3 mentions)
Proflex pcr system, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3d analysis suite, by Thermo Fisher Scientific (2 mentions)
Agpath id one step rt pcr kit, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3d digital pcr, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3d analysissuite cloud software, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d chip reader, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d master mix, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3d system, by Thermo Fisher Scientific (1 mentions)
Proflex 2x flat pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green 1 dye, by Thermo Fisher Scientific (1 mentions)
8 protocols using quantstudio 3d digital pcr chip
1
Digital PCR for Absolute Quantification
2
SARS-CoV-2 sgRNA Detection by Digital PCR
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The presence of sg-RNA was assessed by specific retrotranscription (see [12 (link)]) and digital PCR (dPCR) in 122 longitudinal RNA samples from a sub-cohort of 36 individuals characterized by age, sex distribution and basal viral load compared to those of the whole cohort. The resulting cDNAs were then tested by means of chip-based dPCR using TaqMan assays specific for N and E genes on QuantStudio 3D Digital PCR System (Life Technologies, Carlsbad, CA, USA). In particular, 6 µL of each cDNA was combined with QuantStudio™ 3D Digital PCR Master Mix (Life Technologies) and the TaqMan assay specific for N or E gene and, then, loaded on QuantStudio™ 3D Digital PCR Chips according to manufacturer’s instructions. Each sample was tested in duplicate on two different chips. Loaded samples were then amplified on ProFlex PCR system (Life Technologies) and the end-point fluorescent data were analyzed by QuantStudio™ 3D Digital PCR Instrument (Life Technologies) and the QuantStudio™ 3D AnalysisSuite™ (Life Technologies).
3
Digital RT-PCR for GSDMB Isoform Quantification
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To measure GSDMB Δ5–8 isoform levels and total GSDMB transcript, digital RT-PCR reactions were performed on a QuantStudio 3D Digital PCR System (Thermo Fisher Scientific) using the QuantStudio 3D Digital PCR Master Mix (Thermo Fisher Scientific) and 1 μL of cDNA as template. Custom TaqMan assays were designed to amplify GSDMB Δ5–8 isoform and total GSDMB isoforms. The sequences of primers and probes used in digital RT-PCR assays are listed in Supplementary Table S1 .
Each reaction mixture was loaded onto a QuantStudio 3D Digital PCR Chip (Thermo Fisher Scientific) and cycled for 40 cycles using standard conditions. End-point fluorescence data were analyzed through the QuantStudio 3D Digital PCR Instrument and the QuantStudio 3D Analysis Suite (Thermo Fisher Scientific), according to the manufacturer’s instructions.
Each reaction mixture was loaded onto a QuantStudio 3D Digital PCR Chip (Thermo Fisher Scientific) and cycled for 40 cycles using standard conditions. End-point fluorescence data were analyzed through the QuantStudio 3D Digital PCR Instrument and the QuantStudio 3D Analysis Suite (Thermo Fisher Scientific), according to the manufacturer’s instructions.
4
Digital PCR for IκBα and A20 Genes
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Digital PCR was performed for the IκBα and A20 genes using QuantStudio 3D system (Life Technologies). Prepared samples were loaded into QuantStudio 3D Digital PCR Chip and thermocycled using the ProFlex PCR System (ThermoFisher Scientific) according to the manufacturer’s guidelines. Chip analysis was performed using QuantStudio 3D Digital PCR Instrument and Analysis Suite cloud software. Measurements of the amount of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA were used as a normalization reference for input sample quantity.
5
Digital PCR for Androgen Receptor Copy Number
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AR CN was analyzed using the QuantStudio 3D Digital PCR system (ThermoFisher Scientific). PCR reaction was prepared with 7.5 μl of QuantStudio3D Digital PCR master mix (ThermoFisher Scientific), 0.75 μl of Taqman Copy Number Assay for AR (Assay ID: Hs04107225), 0.75 μl of Taqman Copy Number Reference assay for RNaseP (Assay ID: 4403326) and cfDNA or gDNA (about 5 ng) in a total volume of 15 μl. PCR reaction was loaded onto the QuantStudio 3D Digital PCR Chip (ThermoFisher Scientific) and amplified on ProFlex 2x Flat PCR System (ThermoFisher Scientific). The annealing and extension temperatures were set at 60 °C, and PCR was run for 39 cycles. After PCR amplification, chips were read on the QuantStudio 3D Digital PCR Instrument (ThermoFisher Scientific), and a secondary analysis was performed with QuanStudio 3D Analysis Suit Cloud software (ThermoFisher Scientific). AR CN was calculated using RNaseP as an internal control. The cut-off for indicating a positive AR amplification in cfDNA was AR CN > 1.54 copies/μl, which was the average plus 2 standard deviations of AR CN in cfDNA obtained from healthy males.
6
Quantitative Detection of PEDV by RT-dPCR
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RT-dPCR was used to determine the exact concentration of PEDV. PEDV was amplified using the AgPath-ID One-Step RT-PCR kit (Thermo Fischer Scientific) with the set of primers and probes described on Table 1 (Zhou et al., 2017 (link)). The 15 μL reaction mixture consisted of 7.5 μL of 2× RT-PCR buffer, 0.6 μL of 25× RT-PCR enzyme mix, 800 nM of each primer, 200 nM of probe, 3.63 μL RNase/DNase-free water, and 3 μL of DNA (diluted 4-, 5-, 6- fold). The reaction mixture was then spread over the QuantStudio 3D Digital PCR chip (Thermo Fischer Scientific) and the chips transferred to the QuantStudio 3D Digital PCR thermal cycler. Amplification was performed as follows: PEDV: 10 min at 45 °C, 10 min at 96 °C, 39 cycles of 2 min at 60 °C and 30 s at 98 °C, and a final elongation step for 2 min at 60 °C. Reactions were performed in duplicate, and a non-template control (NTC) was included in each run.
7
Digital PCR Quantification Using SYBR
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Sample preparation, chip loading, and thermal cycling were performed using the standard conditions recommended in the user’s manual. To prepare the digital PCR master mix, SYBR Green I dye (part #, S7567, Thermo Fisher Scientific) was diluted to 20× in TE buffer at pH 8.0. The 20× stock in TE was discarded after use. The reaction mix was prepared by adding: 7.25 μL QuantStudio 3D Digital PCR Master Mix (part #, 4482710, Thermo Fisher Scientific), 1.45 μL 20× SYBR Green I dye in TE buffer (pH 8), 200 nM each of forward and reverse primers, 10–50 ng total DNA sample, and enough water to bring the volume to 14.5 μL. 14.5 μL of reaction mix was loaded onto the QuantStudio 3D Digital PCR Chip (part #, A26316, Thermo Fisher Scientific) using the chip loader, and run using the standard thermal cycling conditions recommended in the user’s manual. The prepared chips were analyzed using the QuantStudio 3D Digital PCR Instrument (part #, 4489084, Thermo Fisher Scientific). SYBR Green I dye was read in the FAM dye channel due to its similar spectral properties.
8
Digital PCR Quantification of SARS-CoV-2 and PEDV
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RT-dPCR was used to determine the exact concentration of SARS-CoV-2 control (nCoV-ALL-Control plasmid, Eurofins Genomics, Germany) and PEDV. Controls were amplified using the AgPath-ID One-Step RT-PCR kit (Thermo Fischer Scientific) with the set of primers and probes described on Supplementary Table S1 (PEDV; E_Sarbeco and RdRP assays (Corman et al., 2020 (link))). The 15 μL reaction mixture consisted of 7.5 μL of 2 × RT-PCR buffer, 0.6 μL of 25 × RT-PCR enzyme mix, 800 nM of each primer, 200 nM of probe, 3.63 μL RNase/DNase-free water, and 3 μL of DNA (diluted 4-, 5-, 6- fold). The reaction mixture was then spread over the QuantStudio 3D Digital PCR chip (Thermo Fischer Scientific) and the chips transferred to the QuantStudio 3D Digital PCR thermal cycler. Amplification was performed as follows: i) SARS-CoV-2: 10 min at 45 °C, 10 min at 96 °C, 39 cycles of 2 min at 58 °C and 30 s at 98 °C, and final elongation step for 2 min at 58 °C; ii) PEDV: 10 min at 45 °C, 10 min at 96 °C, 39 cycles of 2 min at 60 °C and 30 s at 98 °C, and a final elongation step for 2 min at 60 °C. Reactions were performed in duplicate, and a non-template control (NTC) was included in each run.
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