Random primer reverse transcription kit

Following total RNA extraction from the tissues with TRIzol (Sigma-Aldrich, Germany), reverse transcription was performed using a random primer reverse transcription kit (Thermo, United States). The SYBRGREEN kit (TaKaRa, Japan) was applied for detecting respective mRNA expression, with GAPDH or U6 as an internal reference. A total of six replicates were set up for qRT-PCR assay. Quantitative expressions of target genes were calculated using the 2^–△△Ct method. Table 1 presents the primer sequences used.

qRT-PCR was performed to detect the expression levels of Ly6g6e in the spinal cord dorsal horn tissue of rats in different groups. Briefly, total RNA was extracted from the tissues using Trizol reagent (15596018, Thermo Fisher, USA). The concentration and purity of the extracted RNA were assessed using a NanoDrop spectrophotometer. Subsequently, cDNA was synthesized using a random primer reverse transcription kit (4374966, Thermo Fisher, USA). The expression levels of the key genes were determined using the SYBR™ Green PCR premix (4309155, Thermo Fisher, USA), following the manufacturer’s instructions. β-actin was used as an internal reference control. The experimental data obtained from quantitative real-time PCR (qRT-PCR) were analyzed using the 2^–ΔΔCt method to calculate the relative expression of the target gene. The primer sequences used in the qRT-PCR are provided in Supplementary Table 1.

The total RNA of cells in each group was extracted using the TRIzol method. Subsequently, the concentration and purity of RNA were detected by NanoDrop and cDNA was prepared according to the random primer reverse transcription kit (Thermo, USA). The mRNA expression levels of Runx2, OPN, DSPP, and DMP1 were detected according to the instructions of the SYBR GREEN kit (Takara, Japan), and GAPDH was used as the internal reference. Six replicates were set up in the experiment. The relative expression of the target gene was calculated by the 2^−ΔΔCt method. Primer sequences are shown in Table 1.

Total RNA was extracted with TRizol reagent (Invitrogen, USA). After determining the RNA purity and concentration, cDNA was converted following the instruction of random primer reverse transcription kit (Thermo, USA). Then cDNA (2 µl) was used in a 20 µl qPCR reaction including 5 µM of each primer and 10 µl SYBR GREEN PCR Master Mix (TaKaRa, Japan). The reactions were performed on a CFX 96 Touch Real-Time PCR Detection System (Bio-Rad) with the cycling condition: 95°C for 10 min and 40 cycles of 95°C for 10 s, and 58°C for 30 s. GAPDH and U6 were internal reference controls, and this experiment should be repeated for 6 times. Relative gene expression was calculated using the 2^−ΔΔCt method. The primer sequences were designed and synthesized by Sangon Biotech (Shanghai, China), and the primer sequences are shown in Table 1.
Table 1.

Quantitative primers

RNA	Sequences (5ʹ to 3ʹ)
miR-21	F: 5ʹ- GCTTATCAGACTGATGTTG
	R: 5ʹ- GAACATGTCTGCGTATCTC
MMP-2	F: 5ʹ- AGCGAGTGGATGCCGCCTTTAA
	R: 5ʹ- CATTCCAGGCATCTGCGATGAG
MMP-9	F: 5ʹ- GCCACTACTGTGCCTTTGAGTC
	R: 5ʹ- CCCTCAGAGAATCGCCAGTACT
U6	F: 5ʹ- CTCGCTTCGGCAGCACAT
	R: 5ʹ- TTTGCGTGTCATCCTTGCG
GAPDH	F: 5ʹ- GTCTCCTCTGACTTCAACAGCG
	R: 5ʹ- ACCACCCTGTTGCTGTAGCCAA

TRizol method (Solarbio, China) was employed to extract total cellular and tissue RNA, followed by the detection of the concentration and purity of RNA with NanoDrop. Then, RNA was utilized to prepare cDNA according to the instructions of the random primer reverse transcription kit (Thermo, USA). The expression level of circ_0001946, miR-21, GPD1L, and HIF-1α in the cells was measured per the instructions of the SYBR Green kit (TaKaRa, Japan). U6 and GADPH served as internal controls, and six replicates were set up in the experiment. The experimental data obtained by qRT-PCR were used to calculate the relative expression of the target gene with the 2 ˉ^ΔΔCt method. Primer sequences are displayed in Table 1.

The total RNA from cells was extracted with 1 mL TRizol reagent (Invitrogen, USA). Subsequently, NanoDrop was used for determining the concentration and purity of RNA, and cDNA was prepared by the random primer reverse transcription kit (Thermo, USA). 2 µl cDNA was used in a 20 µl qPCR reaction. The miR-214-5p and lncRNA DANCR expression levels were evaluated by the SYBR GREEN kit (TaKaRa, Japan). The cycling condition was: 95°C for 10 min and 40 cycles of 95°C for 10 s, and 58°C for 30 s. GAPDH and U6 were selected as internal reference for lncRNA and miRNA. The qRT-PCR experimental data were counted following the 2^−ΔΔCt method to determine the target gene expression. The following Table 1 include the primer sequences.
Table 1.

Primer sequences

RNA	Sequences(5ʹ to 3ʹ)
miR-214-5p	F: ACACTCCAGCTGGGCGTGTCGTTCACATCT
	R: CUACAAAGGGAAGCGACAGGCA
lncRNA DANCR	F: GCCACAGGAGCTAGAGCAGT
	R: GCAGAGTATTCAGGGTAAGGGT
GAPDH	F: CATCACTGCCACCCAGAAGACTG
	R: ATGCCAGTGAGCTTCCCGTTCAG
U6	F: CTCGCTTCGGCAGCACAT
	R: TTTGCGTGTCATCCTTGCG

The TRizol method was used to extract total RNA from tissues and cells, and a miRNA extraction and isolation kit (Tiangen, China) was used to extract miRNA. After detecting the concentration and purity of RNA, Random Primer Reverse Transcription Kit (Thermo, USA) was used to reversely transfer the mRNA to cDNA, and the miRNA cDNA first-strand synthesis kit (Tiangen, China) was used to synthesize the miRNA cDNA. The expression levels of TCF3, HDAC3, and miR-101 were detected following the instruction of the SYBR GREEN kit (TaKaRa, Japan). GAPDH and U6 were chosen as internal control controls, and the experiment set 6 replicates. The experimental data were dealt with the 2^−ΔΔCt method to calculate the relative expression of target genes. The primer sequences are in Table 1.
Table 1.

Primer sequence

RNA	Sequences(5ʹ- to 3ʹ-)
TCF3	F: 5ʹ- CCAGACCAAACTGCTCATCCTG
	R: 5ʹ- TCGCCGTTTCAAACAGGCTGCT
miR-101	F: 5ʹ- GGGTGGCCATTGCTAATGCT
	R: 5ʹ-GCACTCAGGGTAGGTCAT
HDAC3	F: 5ʹ- GAGTTCTGCTCGCGTTACACAG
	R: 5ʹ- CGTTGACATAGCAGAAGCCAGAG
U6	F: 5ʹ- CTCGCTTCGGCAGCACAT
	R: 5ʹ- TTTGCGTGTCATCCTTGCG
GAPDH	F 5ʹ-CGGAGTCAACGGATTTGGTCGTAT-3’
	R 5ʹ-AGCCTTCTCCATGGTGGTGAAGAC-3’