Sybr green real time pcr master mixture

By using Trizol (Invitrogen (Invitrogen, Carlsbad, CA), total RNA was extracted from cells followed by reverse transcription of RNA to cDNA using the PrimeScript RT reagent Kit (Takara, Shiga, Japan). qRT-PCR was performed with SYBR Green Real-time PCR Master Mixture (Takara, Shiga, Japan) using an Mx3000Ps qRT-PCR system (Applied, Foster City, CA, USA). The relative gene expression was normalized to the internal control GAPDH using the formula 2^-ΔΔCt. The PCR conditions were as follows: pre-denaturing at 95° C for 15s and 45 cycles at 95°C for 5s and 60°C for 30s. The primers used in study were as follows: FLVCR1: 5′- GTGAGATTGGAGGGACAA GTAT-3′(upstream), 5′- TTTCATGGATGAGGAAAACG-3′ (downstream); CSE1L: 5′- CAGAAC ACGCTGACAAGTATCT-3′(upstream), 5′-AGCCCTGCGTCTAGTATCAATA-3′ (downstream); GAPDH:5′-TGACTTCAACAGCGACACCCA-3′ (upstream), 5′- CACCCTGTT GCTGTAGCCA AA-3′(downstream).

After incubation of transfected cell for 48 h, the total RNA was extracted using Trizol reagent (Takara Bio Inc., Japan) based on the manufacturer’s instructions. RNA transcription to cDNA was performed with the help of One-Step SYBR® RT-PCR Kit (Takara Bio Inc., Japan). To do so, 2 µg of total RNA was used. Then, cDNA was mixed with SYBR® Green Real-time PCR master mixture (Takara Bio Inc., Japan) consisted of 10 μM forward primer (0.5 µl), 10 μM reverse primer (0.5 µl), 2X concentration of SYBR Premix Ex Taq (10 µl), and RNase-free water (7 μl). The final mixture was transferred to the Light-Cycler480 real-time PCR system (Roche) to perform the qRT-PCR. Before the main reaction which was conducted using 2 µl of cDNA, a test reaction was carried out using 1 ml of cDNA. The system setting included an initial temperature of 95 °C for 30 s to induce denaturation and activation. Afterwards, intermittent shift of temperature from 95° C for 5 s to 60° C for 30 s was done to allow amplification and quantification for 40 cycles. During the process, melting and standard curves were created to confirm specificity and used to determine the relative amount of desired gene mRNA. Analysis was performed using the delta delta CT method (ΔΔCT Method) based on β-actin mRNA level which is a housekeeping gene. Gene primer sequences are available in Table S2.