Platinum sybr green quantitative pcr super mix udg kit

Total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Complementary DNA was synthesized from the total RNA (0.5 μg) using the Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Madison, WI, USA) following the manufacturer’s instructions. Subsequently, quantitative reverse transcription polymerase chain reaction (RT-PCR) of KLF4 was performed using the Platinum SYBR Green quantitative PCR SuperMix UDG Kit (Invitrogen). Each RT-PCR consisted of 2 μL diluted RT product, 10 μL SYBR Green PCR Master Mix, and 250 nM forward and reverse primers in a total volume of 20 μL. Reactions were carried out on a 7300 RT-PCR System (Applied Biosystems, Foster City, CA, USA) for 40 cycles (95 °C for 5 s, 60 °C for 40 s) after an initial 2-min incubation at 95 °C. As an internal control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene primers were used for RNA template normalization. The relative expression level was calculated using the following equation: relative gene expression = 2 – (ΔCt_Sample – ΔCt_Control) [4] (link). The following primers were used: KLF4, 5′-ATCTTTCTCCACGTTCGCGTCTG-3′ (sense) and 5′-AAGCACTGGGGGAAGTCGCTTC-3′ (antisense); GAPDH, 5′-AAGGTCGGAGTCAACGGATTT-3′ (sense) and 5′-ACCAGAGTTAAAAGCAGCCCTG-3′ (antisense). Experiments were performed at least three times with samples analyzed in triplicate.