Luna universal qpcr master mix protocol

Total RNA was extracted from HGK12 and SAS cell lines co-cultured with F. nucleatum (MOI 1:100) by using TRIzol reagent. As a negative-bacterial control, Fusobacterium mortiferum, a suggested non-invasive and non-pathogenic species within the Fusobacterium genus, was utilized^{73 (link)}. One µg of total RNA was applied for generating complementary DNA by LunaScript® RT SuperMix Kit (NEB, #E3010) followed by standard RT-PCR reaction with Luna® Universal qPCR Master Mix Protocol (NEB, #M3003). The RT-PCR primers used for gene expression validation are listed in Supplementary Table 22.

Relative abundance of each mRNA species was assessed using the Luna^® Universal QPCR Master Mix Protocol (#M3003) (New England Biolabs Ipswich, MA) to perform Q-PCR. Briefly, forward and reverse primers for specific genes, at a final concentration of 0.25 μM, were incubated with 100 ng of template cDNA and 1X Luna Universal qPCR Master Mix for the following steps: 60 s, 95°C for 1 cycle (initial denaturation step), 15 s, 95°C (denaturation step), 30 s, 60°C for 45 cycles (extension step), and 60°C–95°C (melt curve) (New England Biolabs). Differences in threshold cycle number (CT) were used to quantify the relative amount of PCR target contained within each tube. Relative mRNA species expression was quantitated and expressed as transcript accumulation index (TAI = 2−(ΔΔCT)), calculated using the comparative CT method (Bustin, 2002 (link); Radonic et al., 2004 (link); Reynolds et al., 2012a (link); Reynolds et al., 2012b (link)). All values were normalized to the constitutive expression of the housekeeping gene, TFRC (human) or PPIA (mouse). Primers sequences are shown in Table 1.