Thunderbird sybr qpcr mix reagent

Real time-PCR was performed with THUNDERBIRD SYBR qPCR Mix reagent (TOYOBO, QPS-201) in a real-time PCR system (Stratagene). The following primer pairs were used: RAB7 (sense 5‘-GGGGA CTCTGGTGTTGGAA-3′; antisense 5‘-CGCTCCTATTG TGGCTTTGT-3′); RAB5 (sense 5‘-AGCCAGAAGCCAG TGTTGTA-3′; antisense 5‘-GGTTTTTGCCATTCAGGAA GA-3′; and GAPDH (sense 5‘-TCTTTTGCGTCGCCAGCC GAG-3′; antisense 5‘-TCC CGTTCTCAGCCTTGAC GGT-3′).

HepG2 and SH-SY5Y cells lines were obtained from the American Type Culture Collection and Korean Cell Line Bank, respectively. For hypoxia treatment, cells were incubated in a Modular Incubator Chamber (Billups-Rothenberg Inc., CA, USA) filled with 1% oxygen, 5% CO₂, and 94% nitrogen for 18 h. Total RNA was prepared, and cDNA was synthesized using reverse transcriptase. Quantitative PCR was performed using Thunderbird SYBR qPCR Mix reagent (TOYOBO, Japan) and a CFX Connect Real-Time PCR system (BioRad, CA, USA). ACTB was used as an endogenous control. Primer sequences can be found in Supplementary Table S9.

Total RNA was extracted using TRIzol reagent (Thermo Fisher) according to the manufacturer’s protocol. One µg of RNA was reverse-transcribed using the PrimeScript-RT Master Mix (TaKaRa). Real-time qRT-PCR analysis was performed using the Thermal Cycler Dice real time system (TaKaRa) or CFX Connect system (BIO-RAD), together with Thunderbird SYBR qPCR mix reagent (Toyobo). The relative gene expression levels were calculated by normalizing to that of GAPDH. The sequences of primers used for qRT-PCR are listed in Supplementary Table 1.

According to the manufacturer’s instructions, total RNA was isolated from six liver samples of the Nile tilapia at 7 weeks post-feeding using RNAiso reagent (Takara Bio Inc., Japan). A Nanodrop lite spectrophotometer (Thermo Scientific, US) was used to quantify the RNA concentration at OD 260/280 nm. A cDNA was synthesized using SuperScript III First-Strand Synthesis System with Oligo-dT primers (Invitrogen, USA), according to the manufacturer’s instructions. The RT-qPCR reaction was carried out using Step One Plus ™ Real-time PCR machine (Applied Biosystems, USA) to quantify stress (Hsp70, GPx, GST), immune-related genes (IL1-β, TNF-α, TGF-β1, IL-10), apoptotic (caspase3, PCNA), and lipid metabolism-related genes (FAS, PPARα). β-Actin was included as a housekeeping gene. The primer details were previously published [44 (link)–46 (link)]. Per the manufacturer’s procedures, each reaction was conducted in a volume of 20 μl via Thunderbird SYBR qPCR Mix reagents (Toyobo, Japan). The amplification program was 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min with a final dissociation analysis step. After the cycling protocol, the melting curves were obtained to assess the specificity. The mRNA expression data were standardized to the β-Actin using the 2^−ΔΔCT method [47 (link)].

One microgram of total RNA isolated from the renal tissues of WT mice induced by HFD/multiple low-dose STZ and Saa3-promoter luc mice induced with two moderate doses of STZ was reverse transcribed into cDNA using ReverTra Ace (TOYOBO, Osaka, Japan) and random hexamers (TaKaRa Bio, Kyoto, Japan), according to the manufacturer’s instructions. Quantitative PCR reactions were then performed on StepOnePlusTM (Applied Biosystems, Foster City, CA, USA) using THUNDERBIRD SYBR qPCR Mix reagents (TOYOBO) under previously described conditions [13 (link)]. The primers used for the PCR analysis are listed in Table 2. The expression level of the target gene was normalized to that of the housekeeping gene L19. The relative gene expression was calculated using the comparative Ct (2^−ΔΔCt) method.