Proflex 2x flat pcr system

The HER2 amplification status was assessed by the ratio of plasma HER2 copy number to reference gene copy number (HER2 ratio), which was performed on a ProFlex 2X Flat PCR system (Cat No: 4484078, Thermo Fisher Scientific, Waltham, MA, USA) using the HER2 Amplification detection kit (Cat No: Q0137365402, Questgenomics, Nanjing, JS, China). A total 14.5μl dPCR reaction mixture was prepared with 5.8 μl cfDNA sample and RNase-free water (about 5ng cfDNA input), 7.25μl dPCR Master Mix and 1.45μl HER2 amplification detection reaction solution. The dPCR reaction mixture was loaded into chip wells using the Questgenomics Chip Loader and then was sealed and loaded onto ProFlex 2X Flat PCR system according to the manufacturer's instructions. The cycling condition was as follows: 96 °C for 10 minutes, 39 cycles of 60 °C for 2 minutes and 98 °C for 30 seconds, followed by a final extension step at 6 °C for 2 minutes. The chip images were captured with the Questgenomics Biochip Reader and further analyzed using the Cloud Software from Questgenomics. Negative controls with no DNA were included in each run. A HER2 ratio ≥1.3 was defined as positive HER2 amplification, and a HER2 ratio < 1.3 was defined as negative HER2 amplification.

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.

We combined 2,25 μl of RT product (RT or PreAmp product obtained in the previous step) with 3,75 μl nuclease-free H₂O, 7,50 μl QuantStudio™ 3D Digital PCR Master Mix, 0,75 μl of TaqMan MicroRNA Assay-1 (20X) and 0,75 μl TaqMan MicroRNA Assay-2 20X (cel-mir-39-VIC).
To avoid pipetting errors, we prepared a stock solution and we included 10 % excess for volume loss from pipetting. This sample mix was added on each chip and loaded on ProFlex™ 2x Flat PCR System with the following program (Table 1):Table 1

PCR run protocol

Step type	Time	Temperature (°C)
Hold	10 min	96
Cycle (40 cycles)	2 min	56
	30 s	98
Hold	2 min	60
Hold	∞	10

Absolute quantification was determined using QuantStudio 3D Digital PCR System (Thermo Fisher Scientific) and analyzed with QuantStudio 3D AnalysisSuite Cloud Software (Thermo Fisher Scientific).