Sybr green premix ex taqtm kit

Total RNA was extracted using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Total RNA was reverse transcribed into cDNA using the PrimeScript™ RT reagent kit (Takara Bio Inc., Shiga, Japan). The SYBR® green Premix Ex Taq TM kit (Takara Bio Inc., Shiga, Japan) was used for real-time PCR (QPCR) analysis which was performed using an ABI 7500 fast Sequence Detector (Applied Biosystems, Carlsbad, CA, USA). All primers are listed in Table S4.

HepG2 cells cultured in DMEM supplemented with 0 mM, 5.6 mM, or 25 mM glucose were treated with BSA or AGEs for 24 hours. Total RNA was extracted using Trizol (Invitrogen) according to manufacturer's instructions. Total RNA was reverse transcribed to cDNA using the PrimeScriptTM RT reagent Kit (Takara Bio Inc., Japan). Real-time PCR analysis was performed using the SYBR green Premix Ex TaqTM kit (Takara Bio Inc., Japan) according to the manufacturer's instructions. The forward and reverse primers set for ChREBP total were 5′-AACTGGAAGTTCTGGGTGTTC-3′ and 5′-AGGGAGTTCAGGACAGTTGG-3′, respectively. The forward and reverse primers for ChREBP-α were 5′-AGTGCTTGAGCCTGGCCTAC-3′ and 5′-TTGTTCAGGCGGATCTTGTC-3′, respectively. The forward and reverse primers for ChREBP-β were 5′- AGCGGATTCCAGGTGAGG-3′ and 5′-TTGTTCAGGCGGATCTTGTC-3′, respectively. The forward and reverse primers for ACC were 5′-GAAAACATCCCGTACCTTCTTC-3′ and 5′-AAGCCTTCACTGTTCCTTCC-3′, respectively. The forward and reverse primers for SCD1 were 5′- GTCCTTATGACAAGAACATTAGCC-3′ and 5′-AATCAATGAAGAATGTGGTGAAG-3′, respectively. The forward and reverse primers for SLC6A9 were 5′-TCTCCCGCCATCATCTTC-3′ and 5′-TTTTCAAACGCTGGAGGAG-3′, respectively. The forward and reverse primers set for GAPDH were 5′-GAGTCAACGGATTTGGTCGT-3′ and 5′-GACAAGCTTCCCGTTCTCAG-3′, respectively. GAPDH was used as the endogenous control.

To examine the qRT-PCR analysis, total RNA was extracted from frozen fruit tissue using RNAiso-mate Tissue Kit (Tiangen, Beijing, China). The purity and quantity of RNA were assessed by Nanodrop 1000 spectrophotometer (thermoscientic, Beijing, China). The RNA was extracted then reverse transcribed into the first-strand cDNA using a one-step RT-qPCR kit (Takara, Shanghai, China). As per the manufacturer instructions, quantitative RT-PCR (qRT-PCR) was performed using an SYBR green Premix Ex TaqTM kit (Takara) on an ABI 7500 real-time PCR detection system. The expression data were normalized with the tubulin gene as an internal control to investigate the gene expression level [68 (link)]. The sets of all primers used for qRT-PCR analysis were designed on Gen script online software (https://www.genscript.com/tools/, accessed on 4 June 2021) are listed in Supplementary Table S2. The experiments were conducted for three biological and technical replicates and relative expression levels for each gene were evaluated via the 2^−△△CT method [57 (link),69 (link)].

Total RNA was extracted from the cultured cells and tissues using Trizol Reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. For RT-PCR, complementary DNA was synthesized with 1 μg total RNA using Prime Script RT Master Mix (Takara, Japan). To quantify the amount of circRNA, miRNA and mRNA, the real-time PCR analyses were performed using SYBR Green Premix Ex Taq^TM kit (Takara) in Biosystems 7500 Sequence Detection System (Applied Biosystems). All of the reactions were performed in triplicate. After the reactions were completed, the cycle threshold (CT) data were determined using fixed threshold settings, and the mean CT was determined from triplicate PCRs. A comparative CT method was used to compare each condition to the control reactions. In miRNA RT-PCR reaction, U6 was used as an internal control, and the relative level of miRNA normalized with U6 was calculated with the equation 2^−ΔΔCT in which ΔΔCT = (CT_miR-762 − CT_U6)_tumor − (CT_miR-762 − CT_U6)_control. For circRNA and mRNA, GAPDH was used as an internal control. The calculation method is similar to that described above. The primer sequence was listed in Table S1.

The cells were seeded in 6-well plates, and treated with AdBMP9, AdGFP and melatonin alone or in combination for 2 days to extract total cellular RNA and perform reverse transcription. The qPCR reaction was carried out using a SYBR Green Premix Ex TaqTM kit (Takara, Japan). The reaction conditions were as follows: 95 °C for 5 s; 60 °C for 30 s; 40 cycles. GAPDH was used as the internal reference gene, and the relative quantification was performed by the ΔΔCt method (Comparative Delta-delta Ct).

20 mg/ml RNase R (Genesee, Guangzhou, China) was incubated with 10 μg RNA at 37°C for 20 min, and a control group without RNase R treatment was set. Total RNA was extracted from tissues and cell lines by TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and reverse transcription was performed with PrimeScript™ RT reagent Kit (Takara, Dalian, China). Following that, a SYBR Green Premix Ex TaqTM kit (Takara, Kusatsu, Japan) was adopted to perform qRT-PCR, with relative RNA expressions calculated by 2^−ΔΔCt formula, and glycerol aldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were regarded as internal references. Primer sequences are detailed in Table 1.Table 1.

Primer sequences

	Forward	Reverse
circ_0062389	5’- TCTGAGAAGCTGCAGTCCAA-3’	5ʹ- TCTGAGAAGCTGCAGTCCAA-3’
miR-1179	5ʹ-GCGGAAGCATTCTTTCAT-3’	5ʹ- CAAGGGCTCGACTCCTGT-3’
PI4KA	5ʹ-AGCTCCGCAGCACTATCATC −3’	5′-TCCATACACCCCAAGAGTTGTG-3’
HMGB1	5ʹ-GTGAACTGCTGCACGAAGAA-3’	5ʹ- GCCTTTGAAATGTGCTCCCA-3’
U6	5ʹ-GACTATCATATGCTTACCGT-3’	5ʹ-GGGCAGGAAGAGGGCCTAT-3’
GAPDH	5’-CTTTGGTATCGTGGAAGGACTC-3’	5ʹ-GTAGAGGCAGGGATGATGTTCT-3’