Green premix ex taq 2 tli rnaseh plus

Total RNAs were extracted using the RNeasy mini kit (74106, QIAGEN), and reverse transcription reactions were performed using the Prime Script RT reagent Kit with gDNA Eraser (Perfect Real Time) (RR047A, Takara). After mixing the generated cDNA templates with primers/probes and Green® Premix Ex Taq™ II (Tli RNaseH Plus) (RR820B (A × 2), Takara), reactions were performed with the Step One Plus TM Real-Time PCR System (Applied Biosystems).
Mouse GAPDH: Forward, 5′-AGGTCGGTGTGAACGGATTTG-3′,
Reverse, 5′-GGGGTCGTTGATGGCAACA-3′;
Mouse Tap1: Forward, GGACTTGCCTTGTTCCGAGAG,
Reverse, GCTGCCACATAACTGATAGCGA;
Mouse Tap-2: Forward, CTGGCGGACATGGCTTTACTT,
Reverse, CTCCCACTTTTAGCAGTCCCC;
Mouse Tap-bp: Forward, GGCCTGTCTAAGAAACCTGCC.
Reverse, CCACCTTGAAGTATAGCTTTGGG.
Mouse Erap1: Forward, TAATGGAGACTCATTCCCTTGGA.
Reverse, AAAGTCAGAGTGCTGAGGTTTG.

Forty-eight hours after transfection, the total RNA was extracted from each group by using RNA Quick Purification kit (ESscience, China). The concentration and purity of RNA samples were determined by UV spectrophotometry. The total RNA of each group was reversely transcribed to generate cDNA with PrimeScript™ RT Master Mix (Perfect Real Time) (Takara, Japan). Real-time PCR was performed on IκBα and ACTB (internal control) with Green Premix Ex Taq II (Tli RNaseH Plus) (Takara, Japan). The PCR reaction conditions were as follows: predenaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, and annealing at 60°C for 30 seconds, for a total of 40 cycles. Cynomolgus monkey IκBα and ACTB primers were designed and synthesized by Shanghai Biotech Co., Ltd, China, and homology analysis was performed on BLAST. Primer sequence of I-κBα: F:5′-CTGGTGTCGCTCCTGTTGAAGTG-3′, 23 bp; R:5′-TGTCATAGCTCTCCTCATCCTCACTC-3′, 26 bp; internal reference ACTB: F:5′-AGATCAAGATCATTGCTCCTCCTG-3′, 24 bp; R:5′-TCACAGTCCGCCTAGAAGCA-3′, 20 bp. The relative expression level of the IκBα gene mRNA was calculated and analyzed by the 2^−ΔΔCt method.