Tb green premix ex taq 2 master mix

Total RNA was extracted from cells using RNAiso Plus (Takara, Kusatsu, Japan). The M5 miRNA qPCR Assay kit (MF307-01; Mei5bio, Beijing, China) was used to determine the expression of miR-136 after transfection. The TB Green Premix Ex Taq II Master Mix (Takara, Kusatsu, Japan) was used to determine the expression of proliferation and differentiation marker genes. The expression of β-actin was used as an internal reference for coding genes, and U6 was used as an internal reference to evaluate miR-301a levels. All primer sequences used for qRT-PCR analysis are listed in Table 3.

The expression of Atf4, Chac1, Chop, Gclc, Gclm, Hmox1 and Nrf2 (Nfe2l2) genes was monitored by qPCR as previously described [23 (link)] with slight modifications. Briefly, the qPCR reactions were performed in duplicate in a final volume of 20 µL using TB Green PreMix ex Taq II Master Mix (Takara Bio Europe, France) and 200 nM primers (Table 1), in a RotorGene 6000 instrument (Corbett life science, Sydney, Australia). The amplification conditions were 95 °C for 10 min, 40 cycles at 95 °C for 10 s and 60 °C for 50 s. To confirm the absence of non-specific products or primer dimers, a melting curve analysis was performed from 65 to 95 °C at the end of each run, with a slope of 1 °C/s, and 5 s at each temperature. A duplicate non-template control was included for each target as a negative control. Gapdh (glyceraldehydes-3-phosphate dehydrogenase) and Gusb (β-D-glucuronidase) were used as reference genes. The relative expression levels were calculated using the 2^−ΔΔCt method [24 (link)].

Total RNA was extracted from cells using RNAiso Plus (Takara, Kusatsu, Japan). gDNA was extracted using DNAiso Reagent (Takara, Kusatsu, Japan) according to the manufacturer’s instructions. Nuclear and cytoplasmic fractions were extracted using the PARIS kit (Thermo Fisher Scientific, Waltham, MA, USA). The RNA concentration and integrity were determined by spectrophotometry using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 1% agarose gel electrophoresis, respectively. RNA was reverse-transcribed using a PrimeScript RT reagent kit with gDNA Eraser (Perfect Real Time, Takara, Kusatsu, Japan) or miRcute Plus miRNA First-Strand cDNA kit (Tiangen, Beijing, China). qRT-PCR was performed on a Bio-Rad CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) using the TB Green Premix Ex Taq II master mix (Takara, Kusatsu, Japan) or the miRcute Plus miRNA qPCR kit (Tiangen, Beijing, China). Based on the circINSR sequence, divergent and convergent primers were designed to determine its authenticity. The expression of all coding genes was normalized to that of β-actin and U6 small RNA was used as an internal reference to evaluate the level of miR-152. The 2^−ΔΔCt method was used to estimate the relative expression levels. All primer sequences used in this experiment are listed in Table 1 and Table 2.

RNA was extracted from cells using RNAiso Plus (9108; Takara, Otsu, Japan) according to the instructions of the manufacturer. However, gDNA was extracted using DNAiso Reagent (9770; Takara, Otsu, Japan). Nuclear and cytoplasmic fractions were isolated using the PARIS kit (AM1921; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). RNA quality was evaluated using the RNA 6000 Nano LabChip Kit and Bioanalyzer 2100 (Agilent Technologies, Beijing, China), while RNA integrity was confirmed through 1% agarose gel electrophoresis. Subsequently, RNA was reverse transcribed following the instructions of the manufacturer, using either the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time; RR047A; Takara, Otsu, Japan) or miRcute Plus miRNA First-Strand cDNA Kit (KR211; Tiangen, Beijing, China). The qRT-PCR analysis was performed using TB Green Premix Ex Taq II Master Mix (RR820A; Takara, Otsu, Japan) or the miRcute Plus miRNA qPCR kit (FP411; Tiangen, Beijing, China) on a Bio-Rad CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA). Table 3 and Table 4 list the primer sequences employed in this study.

Total RNA was extracted using RNeasy plus kit (Qiagen,, Valencia, CA, USA) and was transcribed to cDNA using Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative PCR was performed using QuantStudio™ 5 Real-Time PCR System (Applied Biosystems) with TB Green® Premix Ex Taq™ II master mix (Takara, Kyoto, Japan). Gene specific primers sequences are listed in supplement Table S1.