Iq supermix

Two different primer-probe combinations targeting both the aroA mutant gene and the xanQ gene (total E. coli) were selected for further duplex probe-based qPCR development (Duplex qPCR A and B). The efficiency, LOQ and LOD of the qPCR reactions were determined in technical triplicates of a tenfold dilution series of both the aroA standard fragment and the xanQ standard fragment (10⁸–10⁰ copies/μl). Each 12 μl qPCR reaction contained: 2μl template DNA, 6 μl 2X IQ Supermix (Biorad); 0.5 μM forward, 0.5 μM reverse xanQ primer or 1 μM reverse aroA primer and 0.2 μM of each probe. Cycling was done on a real-time PCR thermal cycler (Biorad) with the following conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 45 s and 62°C for 1 min, with fluorescence detection of the products at the last step of each cycle.

We used ITS-specific primers and probes described in Schaad et al. [37 (link)] to run real time-PCR. The 20 μL reaction was performed in 0.1 mL strip tubes containing 10 μL 2X IQ Supermix (Biorad, Hercules, CA), 100 nM forward primer, 200 nM reverse primer, 200 nM Taqman probe with dye, 5.8 μL of PCR-grade water and 2 μL of template DNA. The real-time PCR was performed on the Eppendorf Realplex at 95 °C for 3 mins for enzyme activation followed by denaturation at 95 °C for 15 s, and extension and annealing at 62 °C for 1 min. The PCR was run for 40 cycles.

To measure expression of genes of interest, total RNA was extracted from cell lines using a PureLink RNA mini kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol, including treatment of extracted total RNA with DNase treatment. For RNA extraction from mouse tissues, frozen tissue samples were cut and stored for at least 16 h in RNAlater-Ice (Thermo Fisher Scientific, Waltham, MA, USA) at −20 °C. After tissue transition, total RNA was isolated from the tissue using the mirVana miRNA isolation kit following the manufacturer’s protocol. The DNA-free kit was then used to remove the genomic DNA from the extracted total RNA. Once RNA was extracted using either of these kits, 1 μg RNA was reversed transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s protocol. RT-PCR was performed as follows: 10 μL of Master Mix (7.5 μL of 2× iQ Supermix; Bio-Rad, Hercules, CA, USA), 1.75 μL of molecular grade water, and 0.75 μL of 20× TaqMan gene expression assay mix (Applied Biosystems, Foster City, CA, USA) were added to 5 μL of diluted (1:25) cDNA. RT-PCR was performed using a C1000 thermal cycler (Bio-Rad, Hercules, CA, USA). All TaqMan assays used were bought from Applied Biosystems. Cyclophilin A (PPIA) and beta-glucuronidase (GUSB) were used as controls.

Patient-specific DNA qPCR assays were performed using a CFX96 Real-Time PCR Detection System (Bio-Rad). Standard curves using 10-fold dilution series of diagnostic DNA (500 ng) were prepared for each patient, according to Euro-MRD guidelines [18 (link)]. Primers and probes were designed with Primer Express 2.0.0 (Applied Biosystems) and Primer3 input software (version 4.1.0) and are listed in Supplementary Table 2. Each reaction was performed in 25 μL volume consisting of 12.5 μL of 2× iQ Supermix (Bio-Rad), 1.25 μL of 20× primers (500 nmol/L) and probe (200 nmol/L) and 500 ng of DNA (in 5 μL). Thermal cycling conditions consisted of pre-cycling hold for 10 min at 95 °C, ten touchdown cycles from 71 to 61 °C for 30 s at 95 °C, 40 cycles at 95 °C for 15 s and 61 °C for 1 min. All breakpoint assays were performed in triplicate along with PB DNA controls and a no-template control. Assays were optimised to reach high PCR efficiency (slope −3.1 to −3.9) and then tested on serially collected patient samples. MRD data were interpreted according to the standardised guidelines set by the Euro-MRD consortium [18 (link)].