Brilliant 3 ultra fast sybr green qrt pcr master mix

For assay development NKA qRT-PCR was performed in 96 well skirted qPCR plates (20 μl) containing 1× LightCycler®480 RNA Master Hydrolysis Probes, 3.25 mM activator Mn(OAc)₂, 500 nM primers, 200 nM probe and 1 μl plasmid DNA as template on an Agilent Technologies Stratagene MX3005P cycler (version 4.10, build 389). qRT-PCR reactions were ran in triplicate from 10⁸ to 10¹ DNA molecules per reaction as follows: reverse transcription for 3 min at 63 °C, activation for 30 s at 95 °C, followed by 45 cycles consisting of amplification for 5 s at 95 °C and 15 s at 60 °C and a cooling step of 40 s at 40 °C.
For testing the samples of the S0 trials NKA qRT-PCR were conducted using Brilliant III ultra-Fast SYBR® Green qRT-PCR Master Mix (Agilent, Edinburgh, UK). Reactions were performed in a 20 μl volume containing 2× SYBR green qRT-PCR master mix, 500 nm primers and 1 μl RNA as template. RNA samples were grouped into I, M and F point plates and ran alongside two standard curves from 10⁸ to 10¹ RNA molecules/reaction and 4 negative controls (NTCs) per plate. The temperature profile was adapted in the reverse transcription step for 10 min at 50 °C, activation for 3 min at 95 °C, followed by 45 PCR cycles as previously described.

RNAs were extracted from the cell samples using TRIzol™ reagent (Thermo Fisher Scientific). SARS-CoV-2 RNAs in the cell samples were determined using Brilliant III Ultra-Fast SYBR^® Green QRT-PCR Master Mix (Agilent Technologies) and PCR primers (Table S3). The PCR reaction mixture was 10 µL of 2 × SYBR Green qRT-PCR Master Mix, 0.4 µL each of E-forward and E-reverse primers, 0.2 µL of 100 mM DTT, 1 µL of RT/RNase Block, 6 µL of nuclease free water and 2 µL of RNA template. The PCR reaction condition was 50 °C, 10 min; 95 °C, 3 min; and 35 cycles of 95 °C, 5 s and 60 °C, 5 s The fold reduction of the RNAs of the antibody-treated groups compared to the infected cells in medium alone are presented. The EC₅₀ values of the PEN-HuscFvs were calculated by using the GraphPad Prism version 9.3 software (GraphPad Software; San Diego, CA, USA).

Total RNA was isolated using TRIzol (Invitrogen Life Technologies). The concentration of total isolated RNA was measured by using a Microplate Spectrophotometer (Epoch, BioTek). qRT-PCR was used to validate mRNA and miRNA expression changes using the Stratagene Mx3005P real-time PCR system (Agilent Technologies). The reverse transcription reactions were performed using a miRNA 1st-Strand cDNA Synthesis Kit (Agilent Technologies) using 500 ng total RNA for each reaction. qRT-PCR was performed using the Brilliant III Ultra-Fast SYBR® Green QRT-PCR Master Mix (Agilent Technologies) to determine miRNA and mRNA expression, and data were normalized to 18S expression using the 2^-ΔΔCt method. Primer sequence for miR-664 was, 5’-TATTCATTTATCCCCAGCCTACA-3’ (forward primer) and a universal reverse primer. Primers sequences for LIF were, 5’-ACAGAGCCTTTGCGTGAAAC-3’ (forward primer) and 5’-TGGTCCACACCAGCAGATAA-3’ (reverse primer). Primer sequences for NEK7 were, 5’-CACCTGTTCCTCAGTTCCAAC-3’ (forward primer) and 5’-CTCCATCCAAGAGACAGGCTG-3’ (reverse primer).