Sybr green qpcr system

mRNA expressions of AQP4 and AQP9 in the discrete cortex, and mRNA expressions of intercellular cell adhesion molecule-1 (ICAM-1) and nuclear factor-κB (NF-κB) in the hippocampus were determined by qPCR. Total RNA was extracted from the discrete cortex or hippocampus (n=6 rats per group at each time point) using a Takara Genomic DNA Extraction Kit (TaKaRa Bio, Inc., Shiga, Japan) at 6, 24, and 72 h after reperfusion. Extracted total RNA was then reverse transcribed to generate cDNA. The reverse transcription reaction was then amplified using SYBR Green qPCR system (TaKaRa Bio Inc.). The fold change in relative mRNA expression was determined using the 2^−ΔΔC_t method and β-actin as an internal control [19 (link)]. All the forward and reverse primers used in the study were shown in Table 1.

Total RNA was extracted from breast cancer cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. cDNA was synthesized using the PrimeScript RT reagent Kit (Takara Bio, Shiga, Japan) in a total volume of 20 μl according to the manufacturer's instructions. PCR was performed with target-specific primers using the Bio-Rad 7900 Sequence Detection System and the SYBR Green qPCR system (Takara) according to the manufacturer's instructions; β-actin was used as an internal control. Briefly, reactions were performed in triplicate containing 5 μl 2× SYBR Premix II, 2 μl cDNA (corresponding to 50 ng RNA/reaction), and 1 μl 10 mM primers in a final volume of 10 μl. All data were analyzed using the 2^-ΔΔCT method, and mRNA levels were normalized to β-actin. The following primers were used:
β-actin: 5′-AAGAGAGGCATCCTCACCCT-3′
and 5′-TACATGGCTGGGGTGTTGAA-3′.
SREBP1: 5′- ACAGTGACTTCCCTGGCCTAT-3′
and 5′-GCATGGACGGGTACATCTTCAA-3′.
SCD1: 5′-TCTAGCTCCTATACCACCACCA-3′
and 5′-TCGTCTCCAACTTATCTCCTCC-3′
FASN: 5′-AAGGACCTGTCTAGGTTTGATGC-3′
and 5′-TGGCTTCATAGGTGACTTCCA-3′
ACLY: 5′-TCGGCCAAGGCAATTTCAGAG-3′
and 5′-CGAGCATACTTGAACCGATTCT -3′

The mRNA levels of the genes were examined by performing the Real-Time qPCR analysis in accordance with the experimental procedures provided by the previous publications. In brief, the total RNA was extracted from the PC tissues and cells by using the Trizol reagent (Invitrogen, USA). Reverse transcription was achieved by the SuperScriptTM II reverse transcriptase (Invitrogen, USA), and the RNA levels of CDK2, CDK6, Cyclin D1, GAPDH, miR-380-3p, and U6 were determined by using the SYBR Green qPCR system (Takara, Japan). The primer sequences for the associated genes are listed in Table 1.