Quantstudio design analysis desktop software

Total RNA was extracted using TRIzol Reagent (15596026, Invitrogen, USA) according to the manufacturer’s instructions. The quality and quantity of the RNA samples obtained were subjected to spectrophotometric analysis using a bio-photometer (Thermo Scientific™ NanoDrop8000). The RNA was then reverse transcribed into complementary DNA (cDNA) using a Reverse Transcription kit (RR037A, Takara Bio Inc., Japan). Quantitative real-time polymerase chain reaction (qPCR) was performed using a FastStart Universal SYBR Green Master Mix (Rox) system with QuantStudio Design & Analysis Desktop Software (Thermo Fisher Scientific). The primer sequences are shown in Table 3. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was utilized as the internal control.Table 3

Primer sequences used for quantitative real-time PCR analysis

Target gene	Forward sequence (5′−3′)	Reward sequence (5′−3′)
Runx2	CAGTATGAGAGTAGGTGTCCCGC	AAGAGGGGTAAGACTGGTCATAGG
Smad1	CCCCAACAGCAGCTACCCCAACTC	TGGGCCATGGGGTCTTCAGGAG
Sp7	CTGGGAAAAGGAGGCACAAAGA	GGGGAAAGGGTGGGTAGTCATT
Cola1	AGAGGCATAAAGGGTCATCGTG	AGACCGTTGAGTCCATCTTTGC
Gapdh	CTGGAGAAACCTGCCAAGTATG	GGTGGAAGAATGGGAGTTGCT

The total RNA from cells was extracted using TRIzol reagent and the cDNA was synthesized with PrimeScript RT reagent kit (Takara Co. Japan), according to the manufacturer’s instructions. Then real-time quantitative polymerase chain (qRT-PCR) reaction was performed using SYBR Green qPCR Master Mix (Rox). Relative gene expression levels were calculated according to the cycle threshold (Ct) values relative to the endogenous housekeeping control gene (Gapdh) using QuantStudio Design & Analysis Desktop Software (Thermo Fisher Scientific). Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as the internal control. The primer sequences used for the experiment are listed in Table 1.Table 1

Primer sequences utilized for quantitative real-time PCR analysis

Target gene	Forward sequence (5′-3′)	Reverse sequence (5′-3′)
ColI	AAGACGAAGACATCCCACCAATC	CAGATCACGTCATCGCACAACA
BMP2	TATCGCAGGCACTCAGGTCAG	GGGTTGTTTTCCCACTCGTTTC
Runx2	CGCCTCACAAACAACCACAG	ACTGCTTGCAGCCTTAAATGAC
ephrinB2	TATGCAGAACTGCGATTTCCAA	TGGGTATAGTACCAGTCCTTGTC
Gapdh	ACATCGCTCAGACACCATG	TGTAGTTGAGGTCAATGAAGGG