Sensifast syber hi rox kit

The extracted DNA samples were subjected to quantitative PCR for Salmonella Typhimurium quantification from week 1 post infection until sacrifice. The qPCR was performed using SensiFAST SYBER HI-ROX Kit (Bioline, Australia) following the manufacture’s protocol. The qPCR reaction was performed in 72 well rotor-gene disc (Qiagen, Australia). The master mix was prepared and added to the disc through Corbett CAS1200 robot (Corbett Life Science, Australia). The 10 μL final reaction volume contained 5 μL SensiFAST SYBER Hi-Rox mix, 1 μL each of the forward (5′-TTTACCTCAATGGCGGAACC) and reverse (5′-CCCAAAAGCTGGGTTAGCAA) TSR3 primers, 1 μL RNase-free water and 2 μL DNA template. The cycling conditions were initial denaturation at 95 °C for 3 min, then 40 cycles of denaturation at 95 °C for 5 s, annealing and extension at 59 °C and 72 °C for 20 and 30 s, respectively.
A standard curve was constructed by spiking non-infected control faeces with known amount of Salmonella Typhimurium. Briefly, serial ten-fold dilutions of Salmonella Typhimurium grown overnight in LB broth were used to spike the faecal samples. The DNA was extracted from the spiked faecal samples to construct a standard curve and limit of detection was established.

RNA (400 ng) was converted to cDNA using SensiFAST cDNA synthesis kit (Bioline, London, England) as per the manufacturer’s instructions. The reaction included random hexamer primers and anchored oligo dT to ensure unbiased 3′ and 5′ coverage and reverse transcription of all regions. qRT-PCR was run using the SensiFAST SYBER Hi-ROX kit (20 μl reaction, Bioline) with forward and reverse primers according to the manufacturer’s instructions. qRT-PCR was performed in a Bio Rad C1000 Thermal Cycler and quantified by a CFX96 Real-Time PCR Detection System using the following conditions: 95 °C for 30 sec; followed by 40 cycles of 95 °C for 5 s, and 60 °C for 5 s, for plate read. All samples were run in triplicate. Relative expression of target mRNA (Mineralocorticoid Receptor (Nr3c2), Glutamate Ionotropic Receptor NMDA type subunit 2 A (Grin2a), Glutamate Decarboxylase 1 (Gad1), Synaptophysin (Syp)) was normalized to Gapdh (see Supplementary Table S6 for primer sequences) by the 2^−ΔΔc(t) method. qPCR validation correlated 99% with RNAseq findings (Supplementary Fig. S5).