Primescript rt reagent kit for rt pcr

HepG-2 cells (1 × 10⁵) were cultured in the four media conditions mentioned above for 1 h, and an additional fifth group was incubated in Pi-free medium with DOX (1 µM) for 1 h. Then, the Pi concentration was elevated to the normal level (125 mg/L) for an additional 1 h. Total RNA from cells in these plates was isolated using an RNA Purification Kit (Tiangen, Beijing, China) and reverse transcribed using a Prime Script RT Reagent Kit for RT-PCR (Takara, Otsu, Japan). For quantitative PCR, template cDNA, primers, and TaqMan Gene Expression Master Mix were used according to the manufacturer’s instructions. Three replicate wells were tested per group, and the relative amount of each gene was normalized to the amount of β-actin using the calculation method of 2^–ΔΔt and then reported as the fold change at the basal level. The sequences of the primers used for RT-PCR are listed in Supplemenatry Appendix 8.

Total RNA was extracted from the ischemic right liver lobe by using the TRIzon method (Invitrogen, CA, USA). The first strand of cDNA was synthesized by using a PrimeScript RT Reagent kit for RT-PCR (Takara, Otsu, Japan). The primers were used as follows (Table 1): GAPDH was the control with the following primers. All data were calculated according to the 2^−ΔΔCT method: ΔΔCt = (Ct Target−Ct GAPDH) test−(Ct Target – Ct GAPDH) control. Control was defined as 1.0 fold.

Total RNA was prepared from frozen tissue or cells using TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s protocol. Total RNA was quantitated and reverse transcribed with PrimeScript RT-reagent Kit for RT-PCR (Takara Biotechnology Co., Ltd., Dalian, China). Quantitative PCR was performed using the Bio-Rad iQ5 real-time PCR detection system using SYBR Premix Ex TaqTM II (Takara Biotechnology Co., Ltd., Dalian, China). The Bulge-LoopTM miRNA qRT-PCR primer set for miRNAs detection were obtained from Guangzhou RiboBio Co. (Guangzhou, China), and U6 was used as internal control. For adipocyte marker genes, including PPARγ, C/EBPα, FABP4 and LPL, expression was normalized to β-actin. Primers for qPCR were (F forward, R reverse): miR-196a-1 F: 5′-CCGCCTAGGTAGTTTCATGTTGT-3′, R: 5′-AATCCATGAGAGATCCCTACCG-3′. U6 F: 5′-ATTGGAACGATACAGAGAAGATT-3′, R: 5′-GGAACGCTTCACGAATTTG-3′. PPARγ F: 5′-AGGACTACCAAAGTGCCATCAAA-3′, R: 5′-GAGGCTTTATCCCCACAGACAC-3′. C/EBPα F: 5′-TGTTGGGGATTTGAGTCTGTG-3′, R: 5′-GGAAACCTGGCCTGTTGTAAG-3′. FABP4 F: 5′-GAGCACCATAACCTTAGATGGA-3′, R: 5′-AAATTCTGGTAGCCGTGACA-3′. LPL F: 5′-GGAGAGAGGAAGGGAAAACAGAG-3′, R: 5′-AGACCGACCAATAAACTGCAAAG-3′. β-actin F: 5′-GGACTTCGAGCAGGAGATGG-3′ R: 5′-AGGAAGGAGGGCTGGAAGAG-3′.

cDNA was synthesized from 1.0 µg total RNA and digested with DNase I using a PrimeScript RT reagent kit for RT-PCR (Takara, Dalina, China) according to the manufacturer’s instructions. The specific primers for poplar (Table S1) were synthesized by the Sangon Biotechnology Company (Shanghai, China). Real-time quantitative RT-PCR was performed with a SYBR Premix ExTaqTM II kit (Takara) with a Roche LightCycler 480 (Roche, Penzberg, Germany). To quantify the relative expression levels of the target genes, the reference gene β-TUB from P. massoniana was used as the internal control [51 (link)], and the primer sequences were as follows: 5’-GTCGTGAATCATGGCATGGC-3’ (forward); 5’-GCCTCACTATCGGTTTCCCA-3’ (reverse). Fold changes were calculated by the relative expressions of treatments compared to the control. Three independent biological repeats were performed for each selected gene (at least two technical repeats per biological repeat).

Total RNA was extracted using RNAiso Plus reagent (Takara, Japan) and reverse transcribed with a PrimeScript RT reagent kit for RT‐PCR (Takara, Japan), following the manufacturers’ protocols. RT‐PCR was performed using an ABI Prism 7500 Sequence Detector (Applied Biosystems, Foster City, CA). The expression level of target genes was analysed by the relative quantity (RQ) value calculated using the ΔΔCt method. Experiments were performed in triplicates for each sample. Table S1 provides details regarding the primers used in our study.