Realplex4 ep gradient mastercycler

qRT-PCR was performed following the protocol reported by [65 (link)]. Briefly, synthesized cDNA was used as the template and DyNAmo ColorFlash SYBR green qPCR kit (Thermo Scientific, catalog no. F416-L) in a Realplex4 ep gradient Mastercycler (Eppendorf). The annealing temperature was set at 56 °C. The primers used for qRT-PCR analysis are listed in Additional file 9: Table S9. For forward and reverse sequences of few primers, references [65 (link) and 66 (link)] were followed. The 16S rRNA gene was used as the housekeeping gene in this study. Relative gene expression was calculated by the threshold cycle (ΔΔCT) method. Genes with a log₂ fold change of ≥ 1.5 were considered upregulated, and those with a log₂ fold change of ≤ -1.5 were considered downregulated. Experiments were carried out in biological triplicates with statistical analyses (t-test) to validate the fold change in expression of the genes. P < 0.05 was considered statistically significant.

RNA was quantified using a Colibri® microvolume spectrometer (TITERTEK BERTHOLD) and normalized for cDNA synthesis. cDNA synthesis was performed using a Verso cDNA Synthesis Kit (Thermo Scientific™, Cat# AB-1453/A). Quantitative real-time PCR (qRT-PCR) was carried out using synthesized cDNA as a template and a 2X DyNAmo ColorFlash SYBR Green qPCR Kit (Thermo Scientific™) in Realplex4 epgradient Mastercycler (Eppendorf). Primers used for qRT-PCR analysis are listed in Table S1. Relative quantification of gene expression was done via the ^ΔΔCt method. The 16 s rRNA gene was used as the housekeeping gene in our study. Log₂ fold change cutoff ≥1.5 was considered to indicate upregulation, and ≤ −1.5 was considered to indicate downregulation.