Lightcycler 480 system

RNA was extracted by the TRIZOL-chloroform extraction method from cells and used as templates for cDNA synthesis using the first-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed in a LightCycler480 System using a SYBR Premix ExTaq kit (Takara, Shiga, Japan). GAPDH was used as an internal control. Gene-specific primers were purchased from IGE Biotechnology (Guangzhou, China) and listed in Table 4:Table 4

The sequences of primers used for QPCR in this study.

Gene (official symbol)	Primer	Sequence
NPY1R	Forward	TACCAGCGGATCTTCCCCAC
	Reverse	AATTTGTCTTTTTCGCTCCTGC
GREB1	Forward	GGGATCTTGTGAGTAGCACTGT
	Reverse	CATACGGGAAGGAGGTCACG
MYB	Forward	CACAGAACCACACATGCAGC
	Reverse	GCAGAGATGGAGTGGAGTGG
CA8	Forward	CCCAGCTACAGATAGAAGAATTTCG
	Reverse	CACTAAGAGGCTGAGTGGGC
SERPINA3	Forward	ATGGGAGATGCCCTTTGACC
	Reverse	CATGGGCACCATTACCCACT
RBM24	Forward	AGATGCACACGACCCAGAAG
	Reverse	CGATCTCGCCGAAGACCTC
GFRA1	Forward	CTGCGCATTTACTGGAGCAT
	Reverse	GAATGTGCTCCACTTGCTGAA
ESR1	Forward	TGTTGAAACACAAGCGCCAG
	Reverse	GGTTGGCAGCTCTCATGTCT
ANKRD11	Forward	TCCTTCGCACCTTCCAGTTC
	Reverse	GGTTCTTGGCCTTGTGCTTG
HDAC3	Forward	AATGCCTTCAACGTAGGCGA
	Reverse	GGGTTGCTCCTTGCAGAGAT

The total RNA was extracted using TRIzol (Life Technologies, USA) according to the manufacturer's instructions. The reverse transcription was performed using transcriptase (Life Technologies, USA), and the real-time PCR was performed in a LightCycler480 System using a SYBR Premix ExTaq kit (Takara, Shiga, Japan). Bulge-Loop™ miRNA qPCR Primer Sets purchased from RioboBio (Guangzhou, China) were used in the mature miRNA reverse transcription and qPCR. U6 small nuclear RNA was used as an internal control.

Renal tissues were separately collected from the control group, the CI-AKI group, and the rhSTC1 treatment group. mtDNA was extracted and measured as previously described [6 (link)]. Plasma DNA from rats of each group was collected by using the QIAamp DNA Mini and Blood Mini Kit (Qiagen, Germantown, MD), and plasma mtDNA were calculated by quantitative PCR through a LightCycler480 System with SYBR Premix Ex Taq II (Takara, Japan). The relative abundances of plasma mtDNA were indicated as threshold cycles (Tc). And higher Tc represents lower levels of mtDNA.

The total RNA from RAW264.7 cells was extracted using the TRIzol reagent (Thermo Fisher Scientific, Shanghai, China) following the manufacturer’s protocol. According to the manufacturer’s instructions, cDNA was synthesized with the main mixture of ReverTra Ace® qPCR RT Master Mix (Toyobo, Shanghai, China). Real-time PCR was performed on a Roche LightCycler 480 system by using SYBR® Premix Ex Taq™ II (Takara, Dalian, China), and the relative mRNA expression of each gene was normalized to GAPDH.

To validate the results of DEG analysis, qPCR experiments were performed on a Roche LightCycler 480 system using SYBR-GREEN1 fluorescent reagents (TaKaRa, Otsu, Shiga, Japan). All designed primers were synthesized at Beijing Liuhe Huada Genomics Technology Co., Ltd. (Beijing, China) (Table S2). The BmactinA3 gene was used as the endogenous control [52 (link)]. qPCR reactions (25 μL) consisted of a denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 15 s, and 72 °C for 45 s. Three biological replicates were performed for each gene, and relative fold changes were determined using the 2^−ΔΔCt method [53 (link)].

The total RNA was isolated from cultured cells using a TRIzol reagent (Invitrogen) according to the established protocol [21 (link)]. The total RNA (1 μg) was reversely transcribed to cDNA using PrimeScript RT Master Mix (Takara, China). Then, qRT-PCR was carried out in a Roche Light Cycler 480 system with SYBR Premix Ex Taq TM (Takara). The primer sequences are shown in Supplementary Table S3. Relative gene expression levels were calculated by normalization to GAPDH and quantification via the 2^−△△Ct method.

Total RNA was extracted from wheat using Trizol reagent (Thermo Fisher Scientific Life Sciences Solutions, CA, USA). RNA was first purified and then checked for integrity. For RT-qPCR, total RNA was reverse-transcribed to cDNA using the FastQuant RT Kit (Tiangen Biotech Co., Ltd., Beijing, China). RT-qPCR was carried out in a Roche LightCycler480 system using the SYBR Premix Ex Taq kit (Takara Biomedical Technology (Beijing) Co., Ltd, Beijing, China). Reactions were set up using the following thermal cycling profile: 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s, 58 °C for 30 s, and 72 °C for 34 s. Each experiment was replicated three times. The relative expression of the target genes was calculated using the 2^−∆∆CT method, with the wheat Actin gene (TaActin) used as the internal reference gene for the analysis. Primers are listed in Table S4.