Total RNA from each sample was isolated with TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. cDNA was synthesized with M-MuLV transcriptase (Fermentas, Canada) in a 20-µl mixture containing 2 µg of RNA. The sequences of forward and reverse primers are given in Table I . Real-time PCR was then performed using SYBR premix EX Taq II buffer (Takara, Japan) on a LightCycler 480 system (Roche, USA). The amplification was carried out as follows: one cycle of 95°C for 30 s, followed by 40 cycles of 5 s at 95°C for denaturing, 20 s at 60°C for annealing, and 20 s at 75°C for extension. The relative levels of CD36, LOX-1, ACAT1, and ABCG1 were calculated according to the formula 2–ΔΔCt. The data are presented as fold changes in target gene expression normalized to β-actin and relative to the untreated control.
Sybr premix ex taq 2 buffer
Manufactured by Takara Bio
Sourced in Japan
SYBR premix EX Taq II buffer is a ready-to-use solution for real-time PCR amplification. It contains SYBR Green I dye, EX Taq DNA polymerase, and necessary reaction components.
Automatically generated - may contain errors
2 protocols using sybr premix ex taq 2 buffer
1
Quantifying Gene Expression Changes
2
Quantitative Gene Expression Analysis by RT-qPCR
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RNA from all 26 samples was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using 4 μL total RNA, 4 μL 5X PrimeScript buffer, and 12 μL RNasefree dH 2 O in a total volume of 20 μL (TaKaRa, Shiga, Japan). PCR amplification was performed in a 20-μL system containing 2 μL cDNA, 10 μL 2X SYBR Premix EX Taq II buffer (TaKaRa), 0.4 μL Rox Reference Dye, 6 μL RNase-free dH 2 O, and 0.8 μL of each 10 μM primer. The thermal cycling conditions included pre-denaturation at 95°C for 30 s, 40 cycles of denaturation at 95°C for 10 s, and annealing at 60°C for 30 s. β-actin was used as an internal control as previously reported. PCR products were identified using 2% agarose gel electrophoresis. Primer sequences and amplification product sizes are shown in Table 2.
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