Asteponeplus real time pcr system

Total RNA was purified from liver tissues using a Takara MiniBEST Universal RNA Extraction kit, and subjected to RT using PrimeScript RT Master mix. qPCR was performed using SYBR Premix Ex Taq and aStepOnePlus™ Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). GAPDH served as an internal control. Sequences of primers were as follows: Forward, 5′-GACAATGGCTCTGGGCTCTGTA-3′ and reverse, 5′-TTTGGCCCATTCCAACCATTA-3′ for α-SMA; forward, 5′-CCATGGAACCGATCAGTGTG-3′ and reverse, 5′-GCCGACTATGCCAGTCAAGAA-3′ for CXCR4; forward, 5′-CACACGTTGGTACAGAGCTCCAG-3′ and reverse, 5′-TGCAGCCCACAGACCAAATATC-3′ for ERK1; forward, 5′-CTGGACCAGCTCAACCACATTC-3′ and reverse, 5′-AGGTAGTTTCGGGCCTTCATGTTA-3′ for ERK2; forward, 5′- AGCACAGATACCACCAAGACACA-3′ and reverse, 5′-CAGGTCTCGCTTCTTCACACAC-3′ for NF-κBp65; forward, 5′-GCCACAGGATTACAAGAAGC-3′ and reverse, 5′-CCACCAGAGTGAAAAGAACG-3′ for β-catenin; and forward, 5′-AAATGGTGAAGGTCGGTGTGAAC-3′ and reverse, 5′-CAACAATCTCCACTTTGCCACTG-3′ for GAPDH. The details of the thermocycling conditions were as follows: 95°C for 30 sec (initial denaturation), followed by 40 cycles at 95°C for 5 sec (exact denaturation) and 60°C for 30 sec (primer annealing and PCR product elongation). The relative quantity of target genes mRNA was normalized to that of GAPDH using the 2-^ΔΔCq method, as previously described (7 (link)).

We performed qPCR as described in our previous work.^{12 (link)} We homogenized fresh ECT samples in Trizol reagent (Life Technologies,
Cat. No: 15596026), using an Omnitip Tissue homogenizer (USA Scientific, Ocala,
USA; Cat. No. 6615-7273) and isolated total RNAs with the RNeasy Mini Kit
(Qiagen, Valencia, USA; Cat. No. 74104) according to the manufacturer’s
instructions. We measured RNA quality and quantity using the NanoDrop ND-2000
(ThermoFisher Scientific). Both 260/280 and 260/230 ratios of RNA samples were
approximately 2.00. We performed reverse transcription with the SuperScript VILO
cDNA synthesis system (Invitrogen, Cat. No.11754-050). To conduct qPCR, we
applied TaqMan Gene Expression Assays (ThermoFisher Scientific) with a
StepOnePlus Real-time PCR system (Applied Biosystems) and used 18 S rRNA as
endogenous control. All qPCR experiments were performed with 2–3 biological
replicates and technical triplicates for each group.