Quantstudio 3d digital pcr master mix

The digital PCR (dPCR) was performed according to the reference protocol for rare mutation using QuantStudio™ 3D Digital PCR System (ThermoFisher Scientific). We combined ctDNA with nuclease-free H₂O, QuantStudio™ 3D Digital PCR Master Mix, and the ready-to-order TaqMan Assays (20X), ID: AH6R5PH for (BRAF) (ThermoFisher Scientific, Waltham, MA, USA)). Results were analyzed with QuantStudio™ 3D Analysis Suite Cloud Software (ThermoFisher Scientific) and the mean number of copies per µL was calculated.

Up to 5 μL of RT product was added to 7.5 μL QuantStudio 3D Digital PCR Master Mix (Thermo Fisher Scientific), 0.75 μL of TaqMan Assay (20X) (Thermo Fisher Scientific) (see Supplementary Table 3) and the volume made up to 15 μL using nuclease-free H₂O (Supplementary Figure 3). All TaqMan Assays were inventoried and none were custom-made. At least one no template control was used for each TaqMan assay on each PCR run. The reaction mix was applied to each QuantStudio 3D Digital PCR 20K Chip (Applied Biosystems) according to the manufacturer's instructions. The dPCR was run on a GeneAmp PCR System 9700 (Applied Biosystems) with a cycle of 10 min at 96°C, followed by 39 cycles of 60°C for 60 s and 98°C for 30 s, followed by 2 min at 60°C before holding at 10°C. Chips were read, and absolute quantification (copies per μL) determined using the QuantStudio 3D Digital PCR Instrument (Thermo Fisher Scientific).

The copy number concentration of a cDNA target sequence was
determined by dPCR on the Applied Biosystems QuantStudio™ 3D Digital PCR System
(Thermo Fisher Scientific) using the QuantStudio™ 3D Digital PCR MasterMix and
oligonucleotide sequences developed in this work (Table S3). The highest expressor of S100A4 mRNA (sample #2097) was used for “translating”
the sample’s Cq values measured by the various
S100A4 RT-qPCR assays into numbers of
transcript copies.

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.

Total RNA was extracted from 4-day cell cultures using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions, and 1 μg was used to synthesize cDNA using the ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). The resulting cDNA was used for dPCR and RT-PCR. dPCR was performed using the QuantStudio 3D Digital PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and the TaqMan Gene Expression Assay (Hs01574659_m1 for NOS3 and Hs00264901_s1 for TM; Applied Biosystems, Foster City, CA, USA) on a QuantStudio 3D Digital PCR system (Thermo Fisher Scientific). NOS3 and TM expression was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RT-PCR was performed using LightCycler Fast Start DNA Master SYBR Green I (Roche Applied Science, Indianapolis, IN, USA) and Light Cycler primer sets for GAPDH (Search LC GmbH Heidelberg, Germany) on a LightCycler instrument (Roche Applied Science) with associated software.

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.

QuantStudio 3D Chips and the ProFlex PCR System (ThermoFisher) were used to determine gene copy number. cDNA was made using 0.5–3 μg of RNA with restricted or standard dNTPs as described above. Reactions were made with QuantStudio 3D Digital PCR Master Mix (ThermoFisher), 5 μL of cDNA, 250 nM RT PCR primers, and supplemented with 1X SYBR Green 1 dye (ThermoFisher). Digital PCR was performed with a hot start (96 C; 10′), 39 cycles of 60 C (2′) and 98 C (30 s), and 1 cycle of 60 C (2′). Chips were read with the QuantStudio 3D Digital PCR Instrument (ThermoFisher) and analyzed using QuantStudio 3D Analysis Suite Cloud Software (ThermoFisher).