Universal sybr green master mix

Total RNA was extracted using TRIzol according to the manufacturer’s instructions. First-strand cDNA was synthesized using upper standard II (Invitrogen), using 1 µg total RNA for each cDNA synthesis reaction. Real-time PCR was performed using SYBR Green Universal Master Mix (Roche) and a primer mixture. GAPDH was used as the internal reference for mRNA expression. The primers used for real-time qPCR were presented in Table 2.
Table 2

The sequence of the primers used in real-time PCR.

Genes	Forwards Primer (5’-3’)	Reverse Primer (5’-3’)
TET2	GCCACTACCACACCACCACC	GCATCGGAGAAGGGCTGCAT
cGAS	CAGAAAAAGAGCGCCCCGGA	TCTTGGCTTCGTGGAGCAGC
STING	GCCACCCCCTTGCAGACTTT	CCTGGTAGGCAATGAGGCGG
TBK1	CAACCTGGAAGCGGCAGAGT	GCGTCTGCCAGTGATCCACC
GAPDH	CACCATCTTCCAGGAGCGAG	AAATGAGCCCCAGCCTTCTC

Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. For the reverse transcription reaction, TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) was applied. qRT-PCR was carried out in 7500 Fast Real-Time PCR System (Applied Biosystem) by using SYBR Green Universal Master Mix (Roche). Briefly, the following PCR program was performed: 50°C for 2 min and 95°C for 5 min, followed by 40 cycles for 15 s at 95°C and 56°C for 45 s. The sequence information is as follows: PAK4, forward primer: 5′-TCCCCCTGAGCCATTGTG-3′ and reverse primer: 5′-TG ACCTGTCTCCCCATCCA-3′; β-actin, forward primer: 5′-CTA TCGGCAA TGAGCGGTTC-3′ and reverse primer: 5′-GATCTTGATCTTCATGGTGCTAGG-3′. Mature PAK4 mRNA levels in cells were measured by 2^−ΔΔCT method, with β-actin as an internal control.

Total RNA was isolated using TRI reagent and reverse transcribed using random hexamers and Superscript III Reverse Transcriptase (Invitrogen) in a final volume of 20 μl. cDNA was diluted 10-fold, 3 μl of which was used as template for each qRT-PCR. qRT-PCRs were performed using SYBR Green Universal Master Mix (Roche) in a final volume of 20 μl, and each was performed in triplicate, with a minimum of three biological repeats. A dilution standard curve was used to determine the relative expression levels.
Primer Sequences:
AXIN2 F: 5′-GCCGACTTCAAAAGCAAAAT-3′
AXIN2 R: 5′-GTAGGGGTTAACGGGATCGT-3′
BMP2 F: 5′-CCAACACCGTGTGCAGCTTCCA-3′
BMP2 R: 5′-GAAACGTCGTGCTGTTTTCCCAC-3′
BMP4 F: 5′-TGAGGAGCTTCCACCATGA-3′
BMP4 R: 5′-TGCTGAGGTTGAAGACGAAG-3′
BMP7 F: 5′-CGAACGCTTCGATAATGAAA-3′
BMP7 R: 5′-TTCTGGAGTCGAGCAGGAAC-3′
EDA F: 5′-TGGTCTCGCATCACTATGAAC-3′
EDA R: 5′-AATACTCCGAGTGCATTGCAG-3′
EDAR F: 5′-TTCTTTCGAGCCACTGTCCT-3′
EDAR R: 5′-CGAGGTCTGTTTTCCAGCAT-3′
ETV5 F: 5′-AGGCCTGGCTAGCTGAAGCTCA-3′
ETV5 R: 5′-ATCTTGGCTGGAGGTGGGGCAT-3′
FGF20 F: 5′-GCCAAGACCACAGCCTCTT-3′
FGF20 R: 5′-TTCCAAGGTAAAGGCCACTG-3′
GAPDH F: 5′-GACAACTTTGGCATTGTGGA-3′
GAPDH R: 5′-GGCTGTGATGGCATGGAC-3′