After the cells seeded at 5 × 105 cells/well in 6-well tissue culture plates were cultured overnight, 2.5%, 5%, and 10% fermentation broths of F. nucleatum were added, respectively. The total RNA was extracted (Total RNA extraction kit, Magen, Guangzhou, China) 3 h later. A total of 1 µg total RNA was used for reverse-transcription using Fast Quant cDNA first-strand synthesis kit (Tiangen, Beijing, China) after assayed by Nano-drop (Thermo Scientific, Waltham, MA, USA).
Hieff UNICON® qPCR SYBR Green Master Mix (Yeasen Biotechnology, Shanghai, China) was used for quantitative PCR and the program as follows: pre-denaturing: 95 °C, 3 min; a total of 40 cycles of 95 °C 10 s, 60 °C 20 s, and 72 °C 20 s, then melting curve analysis at the end. The relative gene expression was calculated by 2−ΔΔCT method [53 (link)], and GAPDH was used as a reference.
Quantitative PCR gene primers were designed by Primer3 [54 (link)] and synthesized by Tsingke (Shanghai, China). The sequences of primers are listed inTable 1 .
Hieff UNICON® qPCR SYBR Green Master Mix (Yeasen Biotechnology, Shanghai, China) was used for quantitative PCR and the program as follows: pre-denaturing: 95 °C, 3 min; a total of 40 cycles of 95 °C 10 s, 60 °C 20 s, and 72 °C 20 s, then melting curve analysis at the end. The relative gene expression was calculated by 2−ΔΔCT method [53 (link)], and GAPDH was used as a reference.
Quantitative PCR gene primers were designed by Primer3 [54 (link)] and synthesized by Tsingke (Shanghai, China). The sequences of primers are listed in
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