Sybr green 1 kit

Total-RNA from cultured cells was extracted using the TRIzol reagent according to the manufacturer’s instructions. The cDNA synthesis was performed in accordance with the protocol of the Takara Reverse Transcription System for real-time PCR [Takara Biotechnology (Dalian) Co., Ltd., China] with 2 μg RNA and reverse transcription performed with random primers. Real-time PCR primers were designed according to http://www.ncbi.nlm.nih.gov. The sequences of the PCR primers used were as follows: AEG-1, forward 5’-CGGTACCCCGGCTGGGTGAT-3′ and reverse 5’-CTCCTCCG CTTTTTGCGGGC-3′; HIF-1α, forward 5’-GTCGGACAGCCTCACCAAACAGAG C-3’and reverse 5’-GTTAACTTGATCCAAAGCTCTGAG-3′; GAPDH, forward 5’-CGGAGTCAACGGATTTGGTCGTATTGG-3′ and reverse 5’-GCTCCTGGAAGA TGGTGATGGGATTTCC-3′. Real-time PCR analysis was carried out on a LightCycler real-time PCR instrument using SYBR Green I kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. Each reaction was carried out in triplicate. Data were analyzed using the 2^-ΔΔCt method as described elsewhere (Fan et al., 2005 ).

Total RNA was isolated from frozen muscle biopsy tissues using TRIzol reagent (Tiangen Biotech Co., Ltd., Beijing, China), according to the manufacturer’s instructions. The concentration of total RNA was measured by spectrophotometry and reverse-transcribed with an RT-PCR kit (Tiangen Biotech Co., Ltd.). Quantitative PCR was performed using a SYBR Green I kit (Tiangen Biotech Co., Ltd.). Primer sequences were as follows: Aβ forward, 5′-CCAGCCAATACCGAAAATGA-3′ and reverse, 5′-TGATGTTTGTCAGCCCAGAA-3′; and β-actin forward, 5′-CCTGTGCTGCTCACCGAGGC-3′ and reverse, 5′-GACCCCGTCTCTCCGGAGTCCATC-3′. PCR conditions were 50°C for 2 min, 95°C for 10 min and 40 cycles at 95°C for 15 sec and 60°C for 60 sec.

Total RNA was extracted from ten whole embryos or hearts per experimental condition using TRIzol Reagent (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions. cDNA synthesis was conducted using a PrimeScript RT Reagent Kit (Takara, Otsu, Shiga, Japan). RT-qPCR was performed in triplicate using 10-fold diluted cDNA and a SYBR Green I kit (Tiangen, Beijing, China) on a CFX96 real-time PCR machine. The following primers were used for RT-qPCR:
XNkx2.5: forward 5′-GGC TAC CAC CTC CAA GAC G-3′, reverse 5′-GTT GGA GTG GGC AGG GTA AG-3′;
XTnIc: forward 5′-TGA ATC CAC TGG GGC TGT TG-3′, reverse 5′-AGA GTA ACG GCC TTC GAA CA-3′;
MHC-alpha: forward 5′-TCA AGG CTG GTT TGT TGG GT-3′, reverse 5′-AAC CAG AAG GGC ATC TCT GC-3′;
XMLC2: forward 5′-GCG CAA TGG TCT TGC TCT TC-3′, reverse 5′-GAG ATC GTG GAG GGC AAA GT-3′;
GAPDH: forward 5′-TAG TTG GCG TGA ACC ATG AG-3′, reverse 5′-GCC AAA GTT GTC GTT GAT GA-3′.
The primers for gata4 and gata6b were used as described [15 (link)].
The PCR cycling conditions were as follows: 1 cycle at 95 °C for 3 min, 39 cycles at 95 °C for 10 s and 60 °C for 30 s. GAPDH mRNA was used as an endogenous control. The relative quantification was calculated using the 2^-△△ct method.