Sybr green based pcr master mix kit

To evaluate the differentiation of osteoblasts, expression levels of osteogenic genes including runt-related protein 2 (RUNX2), osterix (OSX), osteocalcin (OCN) and osteoprotegerin (OPN) were analyzed. MC3T3-E1 cells were plated in 6-well plates (10 4 cells/well; Corning) into 3 repeat wells and cultured in mineralized medium with 4 different glucose levels for 3 and 7 days. Then, total RNA was extracted using the Trizol reagent (Takara Bio Inc, Shiga, Japan) and cDNA was synthesized from 2 μg of total RNA using PrimeScript RT Reagent Kit (Takara Bio Inc) according to the manufacturer's instructions. Real-time polymerase chain reaction (PCR) was performed using SYBR green-based PCR master mix kit from Takara Bio Inc on a Rotor-Gene 3000 real-time PCR system from Corbett Research (Sydney, Australia). The PCR conditions were as follows: initial denaturation at 95°C for 30 seconds followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Fluorescence signal was monitored at 585 nm during each extension. The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was served as an internal control. The primers used were synthesized commercially from Sangon Biotech, and their sequence and size are given in Table 1.

Total RNA was isolated by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA quantity was measured by the NanoDrop2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Complementary DNA synthesis was performed using the M-MLV reverse transcriptase kit (Invitrogen) following the manufacturer's instructions. qRT-PCR was performed using a SYBR green-based PCR master mix kit (Takara, Shiga, Japan) on a Rotor Gene 3000 RT-PCR system (Corbett Research, Sydney, Australia). The PCR condition was as follows: initial denaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds, and thereafter 60°C for 20 seconds. During each extension the fluorescence signal was monitored at 585 nm. GAPDH and U6 served as an internal control.

To detect the expression of osteogenic markers including bone morphogenetic protein 2 (BMP-2), collagen type I (COL-I), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP) and osteocalcin (OCN), total RNA was extracted using the Trizol reagent (Takara Bio Inc., Japan) and cDNA was synthesized from 2 μg of total RNA using PrimeScript RT Reagent Kit (Takara Bio Inc., Japan) according to the manufacturer"s instructions. Realtime polymerase chain reaction (PCR) was performed using SYBR greenbased PCR master mix kit (Takara Bio Inc., Japan) on a Rotor-Gene 3000 realtime PCR system (Corbett Research Inc., Australian). The PCR conditions were as follows: initial denaturation at 95°C for 30sec followed by 40 cycles, each of 95°C for 5sec and 60°C for 30 sec. Fluorescence signal was monitored at 585nm during each extension. The specific primers were synthesized commercially from Sangon Biotech Inc (China) and shown in Table 1. The gene expression levels were analyzed using the 2 -△△CT method and determined relative to housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The fold change from control values was set at 1-fold, and the fold change relative to the control values was obtained and used to express the change in gene expression.