Sybr green 1 master mix

Complementary DNA was prepared using the Tetro cDNA Synthesis Kit (Bioline, Alexandria), as per manufacturer’s instructions. In brief, the reaction mixture was prepared with 2 μL mRNA, 1 μL random hexamers primer, 1 μL dNTPs 10 mM, 4 μL 5 x RT buffer, 1 μL RNase inhibitor and 10 μL DECP treated water with 1 μL Tetro cDNA RT or 11 μL DECP treated water per sample. A negative RT control was used for each sample. PCR was performed on a Thermal Cycler (Biorad, Gladesville) on the following settings: 25°C for 10 min, 45°C for 30 min, 85°C for 5 min and stored at 4°C.
The qPCR reaction mixture was prepared using the SybrGreen I Master Mix (Roche, North Ryde) as per manufacturer’s instructions. In brief, 2 μL cDNA, 2 μL 5 μM forward and reverse primers, 5 μL SybrGreen 1 Master Mix (Roche, North Ryde) and 1 μL water per sample were added to a LightCycler 480 (Roche, North Ryde) 384-well plate. The qPCR was run using the Sybr Green protocol using the LightCycler 480 (Roche, North Ryde) using the following settings: 94°C for 2 min for 40 cycles, 94°C for 15 s, and 60°C for 1 min to measure primer melting temperature. All data were normalised against both housekeeper genes GAPDH and ACTB2 individually before data were grouped, with a Ct value of >35 being deemed not detected. Primers (Gene Works, Melbourne) used for the study are described in S1A Table.

To assess gene expression, total RNA was extracted from cells or LV walls using the Gene Jet PCR purification Kit (Thermo Scientific) and quantified with a Nano-Drop 8000 spectrophotometer (Thermo Scientific). cDNA was synthesized from 25 ng to 150 ng of total RNAs from the rat heart tissues and rat MSCs, respectively, by using the high-capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR was performed by a 7900HT (Applied Biosystems) with a SYBR Green I master mix (Roche) in following conditions: 95 °C for 10 min followed by 50 cycles at 95 °C for 15 s, 64 °C for 30 s and 72 °C for 30 s. TaqMan primers and probes were purchased from Applied Biosystems. Expression was normalized to ubiquitin C. Relative expression to the Sham group is presented in the figures.