Perfect real time sybr green 2

C2C12 cells were transfected with 5 μg Notch1-ICD domain expressing plasmid (Addgene, #26891). After 48 hours of the transfection, the cells were fixed with methanol-free formaldehyde and used for IP following the previously published method [20 (link)] with anti-Notch1 rabbit monoclonal antibody (D1E11, Cell Signaling Technology, Beverly, MA, USA). Putative Notch1 binding regions for ChIP-qPCR were identified using open data sets with ChIP-Atlas analyzer. ChIP-qPCR was performed with Perfect real-time SYBR green II (Takara).

Total RNA was extracted from specimens using TRI Reagent (Molecular Research Center Inc., Cincinnati, OH, USA) and reverse-transcribed with the PrimeScript RT Master Mix Kit (Takara Bio Inc., Shiga, Japan). Quantitative real-time PCR with total cDNA was performed using Perfect real-time SYBR green II (Takara). PCR amplifications were performed on the Thermal Cycler Dice Real Time System Single at 95°C for 20 s followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. To quantify the relative expression of each gene, the Ct (threshold cycle) values were normalized to that of Gapdh and compared with a calibrator using the ΔΔCt method (ΔΔCt = ΔCt _sample − ΔCt _control). To prevent amplification of contaminating genomic DNA, we designed all primers to span at least one intron. Statistical significance was evaluated by Student’s t-test using JMP software version 10.0.0 (SAS Institute, Cary, NC, USA). Primer sequences are listed in Table 1.

Total RNA was collected from MSCs using TRI Reagent® (Molecular Research Center Inc., Cincinnati, OH, USA) and reverse-transcribed with the PrimeScript® RT Master Mix Kit (TAKARA Bio Inc., Shiga, Japan). rRT-PCR was performed by using Perfect real-time SYBR green II (TAKARA). To prevent amplification of contaminating genomic DNA, we designed all primers to span at least one intron.

Total RNA was extracted with the TRIzol reagent (Invitrogen) and reverse transcribed with the High Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). The qRT-PCR with total cDNA was performed using Perfect real-time SYBR green II (Takara Bio, Inc., Shiga, Japan) with appropriate primer sets (Table 2) in the Thermal Cycler Dice® Real Time System (Takara Bio, Inc.) at 95 °C for 20 s followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s. To quantify the relative expression of each gene, the Ct (threshold cycle) values were normalized to an endogenous reference (ΔCt = Ct_target − Ct_Gapdh) and compared with a calibrator (control), using the ΔΔCt method (ΔΔCt = ΔCt_sample − ΔCt_calibrator).

Total RNA was collected using a TRI Reagent® (Molecular Research Center, Inc., OH, USA) and a PrimeScript® RT Master Mix Kit (TAKARA Bio Inc., Shiga, Japan). Quantitative RT-PCR for total cDNA was performed using Perfect real-time SYBR green II (Takara). PCR amplifications were performed using a Thermal Cycler Dice® Real Time System Single at 95 °C for 20s followed by 40 cycles of 95 °C for 5s and 60 °C for 30s. To quantify the relative expression of each gene, the Ct (threshold cycle) values were normalized by Lamina (ΔCt = Ct_target − Ct_Lamina) and compared with a calibrator using the “ΔΔCt method” (ΔΔCt = ΔCt_sample − ΔCt_calibrator). All values are means ± SD of 3 experiments. Statistical significance was evaluated with JMP software version 10.0.0 (SAS Institute, Cary, NC, USA) using either Student’s t-test or the Tukey-Kramer HSD test. The primer sequences are given in Supplementary Table S1.

RNA isolation and subsequent reverse-transcription were performed using TRI Reagent® and the PrimeScript® RT Master Mix Kit (TAKARA Bio Inc.). Quantitative real-time PCR was performed using Perfect real-time SYBR green II (TAKARA Bio Inc.) and a Thermal Cycler Dice® Real Time System. Expression quantity was evaluated by the ΔΔCt method using Gapdh as a control gene for normalization. To prevent contamination of genomic DNA, primers were designed to span at least one intron. Primer sequences are listed in Supplementary Table S2.