Tb premix ex taq 2 kit

Total RNA was extracted from cells with the TRIzol reagent™ (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized using PrimeScript™ RT Master Mix (Takara, Cat# RR036A). RT‐qPCR was performed by TB Premix Ex Taq™ II kit (Takara, Cat# RR820A) and then detected on the CFX96 Real‐Time System (Bio‐Rad). The mRNA levels were calculated using 2‐ΔΔCt method after normalization to the expression of β‐actin. The primers were described in Table S1

Five µl of diluted cDNA samples were amplified in triplicate using TB Premix Ex Taq™ II kit (Takara, Japan) and the CFX96 Touch two-color Real-Time PCR detection System (Bio-Rad, Hercules, USA). The amplification conditions were as follows: activation at 95 °C for 30 s, followed by 41 cycles of denaturation at 95 °C for 5 s, and extension at 60 °C for 30 s. A melting curve analysis was subsequently performed which began at 55 °C for 5 s and increased to 95 °C in 0.5 °C increments. The relative expression levels of each cytokine were then normalized to those of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) following the 2-ΔΔCt method [19 (link)].
To determine the total bacterial load, 100 ng of each DNA sample was used to amplify the 16S ribosomal RNA (16S rRNA) gene. The amplification conditions were the same as described above, except that the extension temperature was 58 °C. Quantification of the total bacterial load from the DNA samples was calculated using the standard curve generated from serial dilutions of known concentration of genomic DNA isolated from a pure culture of Pg and the results were reported as log(copies/μl).
All primer sequences used in this study are listed in Additional file 1: Table S1.