Sybr green mix

Mouse kidney tissues were homogenized in Trizol reagent (TianGen). Total RNA was extracted and reverse transcribed into cDNA (HiScriptIII RT SuperMix, Vazyme). Real-time PCR was performed on LightCycler480 apparatus (Roche) using SYBR Green Mix (Yeasen). Mouse gapdh was used as internal control gene. The relative gene expression was analyzed using 2^−ΔΔCT method. Primers were listed: tcf21-F, cgctcacttaaggcagatcc; tcf21-R, gtcaccacttccttcaggtca; dach1-F, cctgggaaacccgtgtactc; dach1-R, agatccaccattttgcactcatt; ddx17-F, gatcgggatcgtgacaggga; ddx17-R, agtcagtcttgctacttctggat; gapdh-F, tggccttccgtgttcctac; gapdh-R, gagttgctgttgaagtcgca.

Three biological replicates (plant samples) and three technical replicates (qPCR reactions) were used in expression analysis. In detail, for different tissues and elicitor treatments, corresponding samples were collected from three independent plants as biological replicates. In the qPCR assay, each reaction well was replicated three times (they contained the same cDNA template and primers), as technical replicates. The qPCR was conducted using a StepOne Plus instrument (Applied Biosystems, Waltham, MA, USA). The SYBR Green Mix (11184; Yeasen Biotech, Shanghai, China) and an internal control gene TUA (alpha-tubulin) were employed in the qPCR reactions (Zhu et al., 2019 (link)). The specific primers are shown in Table S4. Each PCR mixture (10 μL) consisted of 1 μL cDNA (20× dilution), 5 μL of SYBR Green Mix, 0.4 μL of each primer (10 μM), and 3.2 μL of ddH2O. The amplification was conducted as described previously (Zhu et al., 2021 (link)). The relative expression was computed by the 2^−ΔΔCt method (Livak & Schmittgen, 2001 (link)).

RNA was isolated from MD-MBA-453 cells by Trizol (Thermo), and cDNA was generated by reverse transcription kit (TaKaRa, China). 36B4 (human) was selected as an internal control. Real time QPCR was processed by the SYBR Green Mix (Yeasen, China). Data was acquired and analyzed by StepOnePlus Real-Time PCR System (Thermo).

TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China) was utilized to isolate total RNA from fresh CCA and paracancerous tissues and 6 cell lines. SYBR-Green Mix (Yeasen, Shanghai, China) was used according to the manufacturer’s protocol. Primer (Sangon Biotech, China) information is shown in Supplementary Table S1. B-ACTIN was utilized as an internal control. The 2-ΔΔCt method was applied to calculate the relative expression levels of genes.

Total RNA was extracted from liver tissues or AML12 cells after treatment as stipulated using a RNAfast200 kit (Fastagen, Shanghai, China), and cDNA was synthesized using the PrimeScript RT reagent kit (Takara, Tokyo, Japan). The expression levels of genes of interest and of the HPRT control were assessed by PCR with SYBR Green mix (Yeasen). Transcript levels of target genes were calculated as the ratio of target gene expression to HPRT expression. Fold changes in target gene expression were analyzed by StepOne software v2.3 (Applied Biosystems, Waltham, MA, USA) using the delta/delta CT method. The sequences for the primers were Zc3h12a (F: 5′-ACGAAGCCTGTCCAAGAATCC-3′, R: 5′-TAGGGGCCTCTTTAGCCACA-3′), Atf-3 (F: 5′-GAGGATTTTGCTAACCTGACACC-3′, R: 5′-TTGACGGTAACTGACTCCAGC-3′), Csrnp-1 (F: 5′-CCGTCTACTATTTCCCACGGT-3′, R: 5′-AACTCAGCTAAGGAGAAAAGGC-3′), Irf-1 (F: 5′-ATGCCAATCACTCGAATGCG-3′, R: 5′-TTGTATCGGCCTGTGTGAATG-3′), KIF-11 (F: 5′-CATGGACATTTGTGAGTCGATCC-3′, R: 5′-CCTTTGGTAGATCAGGTGCAG-3′), Ddit-4 (F: 5′-CAAGGCAAGAGCTGCCATAG-3′, R: 5′-CCGGTACTTAGCGTCAGGG-3′).

The sample RNA was extracted using TRizol reagent (Thermo Fisher Scientific, Waltham, MA). Subsequently, the concentration and purity of the extracted RNA were tested by the NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). We took 1 microgram of the above product for reverse transcription process and used the SYBR Green mix (YEASEN, China) to perform PCR by StepOnePlus Real‐Time PCR System (Thermo Fisher Scientific). MicroRNAs primers were obtained from RiboBio, and gene primers are as follows:

Table B: List of primers for PCR

Primer	Sequence
GAPDH
Forward	5′‐GAGTCAACGGATTTGGTCGT‐3′
Reverse	5′‐GACAAGCTTCCCGTTCTCAG‐3
NNT
Forward	5′‐ GTCTCCTGAAATCTGCCCCT ‐3′
Reverse	5′‐ CAGCACAGTGATAACGACGG ‐3′
PGC1A
Forward	5′‐ AGCCTCTTTGCCCAGATCTT ‐3′
Reverse	5′‐ GGCAATCCGTCTTCATCCAC ‐3′
UCP1
Forward	5′‐ GCGGATGAAACTCTACAGCG ‐3′
Reverse	5′‐ TTGATTCCGTGGAGATGGCT ‐3′
CIDEA
Forward	5′‐ TCTGGTGCTGGAGGAAGATG ‐3′
Reverse	5′‐ TGACTCTCGCTATTCCCGAC‐3′
DIO2
Forward	5′‐ TCTCCAACTGCCTCTTCCTG ‐3′
Reverse	5′‐ ACCATTGCCACTGTTGTCAC ‐3′

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