Ssofast evagreen supermix with low rox

Quantitative real‐time PCR (qPCR) assays were performed with AB 7500 (Applied Biosystem).
Samples before the concentration process (called “PRE”) and after (called “POST”) were tested. Dilutions were used.
qPCR conditions included an initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing‐elongation for 1 min. Real‐time PCR was set up with 2X SsoFast EvaGreen Supermix with Low ROX [Bio‐Rad S.r.l., Segrate (MI), Italy] in which EvaGreen was used as a detecting dye; a 10 μl reaction consisted of 5.0 μl SsoFast EvaGreen Supermix with Low ROX, 0.1 μl each 10 μmol/L primer solution, 2 μl DNA sample, and 2.8 μl of Milli‐Q water. Primer sequences, targets, annealing temperatures, and references are given in Table S4.
Standard curves were generated using 10‐fold serial dilutions of positive controls and qPCR amplification efficiencies (E) were based on the following Equation (1):
$E={10}^{(-1/\text{slope})}-1,$
and R² values (linearity) were 0.99.
All samples and standards were run in triplicate.
Negative controls were also tested in triplicate too for each amplification. All the assays were followed by a dissociation stage and melting curves were obtained.
Amplification data were collected and analyzed with the SDS 7500 Real‐Time PCR System Software (Applied Biosystems).

Biomark^™ HD with a 96.96 IFC was used for the RT-qPCR amplification, as previously described [17 (link)]. Briefly, for each sample, a 6 µL sample mix containing 3 µL of 2× SsoFast EvaGreen Supermix with low ROX (Bio-Rad Laboratories, Hercules, CA, USA), 0.3 µL of 20× DNA Binding Dye (Fluidigm), and 2.7 µL of the pre-amplified sample was prepared. A primer stock (100 μM combined forward and reverse primers) was prepared for each assay, and 0.3 µL of the stock was mixed with 3 µL of 2× Assay Loading Reagent (Fluidigm) and 2.7 µL 1× DNA Suspension Buffer to make assay mixes. Finally, 5 μL of each assay and sample mix was transferred into the appropriate inlets according to Fluidigm’s recommendation. After loading, the array was placed in the Biomark HD instrument for quantification and detection using the GE Fast 96 × 96 PCR + Melt v2.pcl PCR thermal protocol. The data were analyzed with Real-Time PCR Analysis Software (Fluidigm) according to Fluidigm’s recommendation. A non-template cDNA/pre-amplification and a non-template pre-amplification control (H₂O) were included and were finally defined as those with Ct values ≥ 35 or that were undetermined.

Glioblastoma tissues were obtained from the operating room and immediately dissociated using a papain-based dissociation system (Worthington Biochemical). Leukocytes were depleted using anti-human CD45 MicroBeads (Miltenyi Biotec). The C1 single-cell auto prep instrument (Fluidigm) was used for capturing single cells. Pre-amplified cDNA was utilized for qPCR with the Biomark HD system with IFC Controller HX (Fluidigm) and 2× SsoFAST EvaGreen supermix with low ROX (Bio-Rad).