Sybr premix ex kit

Total RNAs of HOKs and oral epithelia were extracted with TRIzol reagent (Invitrogen). First-strand cDNA templates were synthesized using PrimeScript RT reagent kit (TaKaRa) in the light of manufacturer’s instructions. Quantitative PCR was conducted using SYBR Premix Ex kit (TaKaRa) according to manufacturer’s instructions. Relative amount of transcripts was calculated according to the 2^-ΔΔCt formula. Sequences of PCR primers were provided in Table 1.Table 1

Primer sequences involved in this work

Primer name	Forward(5′-3′)	Reverse(5′-3′)
hHIF-1α	GAACGTCGAAAAGAAAAGTCTCG	CCTTATCAAGATGCGAACTCACA
hTNFα	CGAGTGACAAGCCTGTAGC	GGTGTGGGTGAG-GAGCACAT
hIL-1β	GCTGAGGAAGATGCTGGTTC	GTGATCGTACAGGTCGCATCG
hIFNγ	TGAACATGATGGATCGTTGG	CATTCACTTTGCTGGCAGTG
hIL-6	AAATGCCAGCCT-GCTGACGAAC	AACAACAATCTGAGGTGCCCATGCTAC
hTREM-1	GGCAGATAATAAGGGACGGAGAG	CATTCGGACGCGCAGTAAA
hGADPH	ACCACAGTCCATGCCATCAC	TCCACCACCCTGTTGCTGTA
mIL-1β	CAGGATGAGGACATGAGCACC	CTCTGCAGACTCAAACTCCAC
mIFNγ	CGGCACAGTCATTGAAAGCCTA	GTTGCTGATGGCCTGATTGTC
mGADPH	TGTGTCCGTCGTGGATCTGA	CCTGCTTCACCACCTTCTTGA
HIF-1α κB ChIP	GACAAGCCACCTGAGGAGAG	CACGCGGAGAAGAGAAGGAA

Trizol (Invitrogen) was used to extract total RNA and PrimeScript RT reagent kits (TaKaRa, Mountain View, CA) were used to reverse transcribe the RNA samples. The qPCR assays were performed with SYBR Premix Ex Kit (TaKaRa) and a Bio-Rad IQ5 system as previously published using the primers described in Supplemental Table I (14 (link)).

Total RNAs from human tissues or HOKs were harvested using TRIzol (Invitrogen, Waltham, MA). The PrimeScript RT Reagent Kit (TaKaRa, Dalian, China) was used to synthesize the first strand cDNAs. The SYBR Premix Ex Kit (TaKaRa) was selected to perform qPCR in a real-time PCR system (Roche480). For miRNAs analyses, the miRNA isolation Kit (QIAGEN, Hilden, Germany) was chosen to extract miRNAs from human samples or cells. The specific miRNA First-strand cDNA Synthesis Kit (Aidlab Biotechnologies, Beijing, China) and a miRNA real-time PCR Assay Kit (Aidlab Biotechnologies) were applied to carry out the first strand cDNAs synthesis and qPCR, respectively. The relative amounts of transcripts were calculated using the 2^-ΔΔCt formula. GAPDH or U6 were regarded as internal control for mRNA or miRNA examinations. The equal amount of exogenous cel-miR-39 was served as internal control for circulating miRNAs in human serum and saliva. The primers for qPCR were listed in Supplementary Table 2.

TRIzol reagent (Invitrogen, USA) was used to isolate total RNAs. Real-time PCR was operated in a real-time system (Bio-RAD IQ5) with SYBR Premix Ex kit (TaKaRa, Japan). Relative amount of transcripts was calculated by the 2^−ΔΔCt formula. PCR primers are displayed in Table 2.Table 2

Primer sequences used in this work.

Primer name	Forward (5′-3′)	Revere (5′-3′)
hsa-mir-346	TGTCTGCCCGCATGCCTGCCTCT
PUMA κB ChIP	CATGTAAGTGATGTCATATGTC	CTTCCTGGTCTTTTCCAAACT

Total RNA was extracted using TRIzol lysis buffer (Invitrogen, Carlsbad, United States), and the concentration of RNA was determined. A PrimeScript RT reagent kit (TaKaRa, Mountain View, CA, USA) was used to reverse transcribe the RNA samples. A SYBR Premix Ex Kit (TaKaRa, Japan) was used with a Bio-RAD iQ5 real-time PCR detection system to perform PCR as previously described [13 (link)]. The primers used in this study are listed in Supplemental Table I.