Lightcycler analysis software

Real-time polymerase chain reaction (PCR) was conducted to quantify the amounts of three periodontopathic bacteria (P. gingivalis, T. denticola, and T. forsythia) using a LightCycler^® system (Roche Molecular Biochemicals, Mannheim, Germany) and Light-Cycler^® DNA Master SYBR Green I (Roche Molecular Biochemicals, Mannheim, Germany). Sequences of species-specific primers were used based on the 16s rRNA gene as previously described [19 (link)] (Supplemental Table S1). Amplification was performed in a 20 μL final volume containing 2 μL of template DNA, 2 μL of PCR Master Mix, 1 μM of each primer, and 4 mM of magnesium chloride. Detailed real-time PCR settings were previously provided by Chigasaki et al. [16 (link)]. Bacterial counts were collected from the stimulated saliva volume, and data were analyzed using LightCycler^® analysis software (Roche); the cutoff value for positivity was set at 1000 counts/mL per sample.

Ishikawa cells were seeded at a density of 10,000 cells per well in a six-well plate overnight. Afterward, the cells were incubated with the medium containing DMEM and 0, 25, 50, and 75 µmol/L kaempferol [22 (link)]. Total RNA was collected using a RNeasy mini kit (RNA Fast 200, China), according to the manufacturer’s instructions. Then, cDNA was synthesized from 1 μg of total RNA using a SureScript First-Strand cDNA Synthesis Kit (GeneCopoeia, USA). Finally, the expression levels of GAPDH, PTEN, MMP1, and MMP9 were measured using an all-in-one qRT-PCR detection kit (GeneCopoeia, USA). The sequences of the primers were as follows: PTEN, 5′-CAAGATGATGTTTGAAACTATTCCAATG-3′ (sense) and 5′-CCTTTAGCTGGCAGACCACAA-3′ (antisense); MMP1, 5′-CACAAACCCCAAAAGCGTGT-3′ (sense) and 5′-TCGGCAAATTCGTAAGCAGC-3′ (antisense); MMP9, 5′-TCTGCCCCGGACCAAGGATA-3ʹ (sense) and 5′-ACATAGGGTACATGAGCGCC-3′ (antisense); and GADPH, 5′-GCACCGTCAAGGCTGAGAAC-3′ (sense) and 5′-TGGTGAAGACGCCAGTGGA-3′ (antisense). Melting curve analysis was used to confirm the amplification specificity. The quantification data were analyzed using LightCycler analysis software (version 4.0; Roche Applied Science, Mannheim, Germany). Relative expression was normalized to that of GAPDH.

The qRT-PCR was performed as described previously [59 (link)] with a LightCycler system (Roche Diagnostics, Lewes, East Sussex, UK). The primers used were from Fasmac (Kanagawa, Japan): TNF, 5′-TCCTTCAGACACCCTCAACC-3′ (forward) and 5′-AGGCCCCAGTTTGAATTCTT-3′ (reverse); TNFR1, 5′-ACCAGGCCGTGATCTCTATG-3′ (forward) and 5′-CAGCTATGGCCTCTCACTCC-3′ (reverse); and TRADD, 5′-GCTTTGGAGATCAGCCTCAC-3′ (forward) and 5′-GTATCTGCAGCACCCAGGAT-3′ (reverse). Human β-actin (Fasmac) was amplified according to the manufacturer’s protocol as an internal control to allow for the quantitation of TNF, TNFR1, and TRADD amplification products. Data were analyzed using the LightCycler analysis software (Roche Diagnostics), and a standard curve correlating cycle number with the amount of products formed was plotted for each sequence of interest. The mRNA expression levels of TNF, TNFR1, and TRADD were then normalized to that of β-actin.

RNA in cultured cells was extracted using an RNeasy Mini Kit (QIAGEN, Tokyo, Japan), and cDNA was produced using PrimeScript RT Master Mix (Takara, Shiga, Japan). The sequences of PCR primers for mRNA in DPD, Sp1, and β-actin were as follows: DPD forward, 5’-GTTGTGGCTATGATTGATGA-3’, and reverse, 5’-ATTCACAGATAAGGGTACGC-3’; Sp1 forward, 5’-TTGAAAAAGGAGTTGGTGGC-3’, and reverse, 5’-TGCTGGTTCTGTAAGTTGGG-3’; β-actin forward, 5’-GCAAAGACCTGTACGCCAAC-3’, and reverse, 5’-CTAGAAGCATTTGCGGTGGA-3’. PCR reactions were performed with 20 ng of cDNA with LightCycler 480 SYBR Green I Master (Roche Molecular Biochemicals, Indianapolis, IN, USA). Quantitative RT-PCR was performed on a Roche LightCycler 480 system (Roche Molecular Biochemicals). Quantification data were analyzed with the LightCycler analysis software (Roche Molecular Biochemicals).

Reverse transcription was performed as described71 (link). Total RNA was DNase treated prior to reverse transcription to remove genomic DNA. Reverse transcription reactions were verified by Β-actin RT-PCR using cDNA amplified with GoTaq Flexi (Promega; Madison, WI, USA). QPCR was performed using SYBR Green GoTaq qPCR master mix (Promega) according to manufacturer’s instructions on LightCycler 96 SW 1.0 (Roche; Castle Hill, NSW, Australia). Primer sequences have been supplied (S1 Table). Reactions were performed on cDNA equivalent to 50 ng of total RNA and carried out for 45 amplification cycles. SYBR Green fluorescence was measured after the extension step at the end of each amplification cycle and quantified using LightCycler Analysis Software (Roche).