RNA Extraction and Reverse Transcription Polymerase Chain Reaction

Total RNA was extracted from fresh gingival tissues (n=8) and cultured HGFs (n=4) with TRIzol regent (Life Technologies, Carlsbad, CA, USA) and MiniBest Universal RNA Extraction Kit (TAKARA, Tokyo, Japan), respectively, according to the manufacturer’s instructions. The precipitate was air-dried and resuspended in RNase-free water. The purity and concentration of isolated RNA were determined by a nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was generated from a total of 1 µg RNA using RT reagent Kit with gDNA Eraser (TAKARA, Tokyo, Japan). Aliquot without reverse transcriptase was prepared as the negative control. PCR was performed using PrimeSTAR^® Max DNA Polymerase (TAKARA, Tokyo, Japan) to detect the existence of TAS2Rs and downstream molecules (primers listed in Supplementary Table 1 ). Besides, quantitative real-time PCR was performed to determine the relative expression level of TAS2Rs in HGFs (primers listed in Supplementary Table 1 ). The expression level of target genes normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was calculated by 2^-ΔΔCT method.