Mirna qpcr detection kit

Total RNA was extracted using an RNA extraction kit (Tiangen Biotech, Beijing, China). For mRNA detection, the SuperScript IV reverse transcriptase system (Thermo Fisher Scientific, Carlsbad, CA, USA) was used for cDNA synthesis. PCR reactions were performed using the SYBR® Premix Ex Taq™ (Takara, Dalian, China). For miRNA detection, RNA was reverse transcribed into cDNA by using the miRNA First-Strand cDNA synthesis kit (Tiangen). PCR was conducted using the miRNA qPCR detection kit (Tiangen). Data were processed with the 2^−ΔΔCt method after normalizing to GAPDH or U6. Primer sequences are given in Additional file 1: Table S2.

TRIzol Regent was used for total RNA extraction of patient samples and cell lines (Invitrogen). RNA quality was tested by NanoDrop 2000 (Thermo Scientific). FastQuant RT Kit or miRNA RT Kit was used for reverse transcription (TIANGEN). Super‐Real PreMix Plus Kit or the miRNA qPCR Detection Kit (TIANGEN) was used to analyse target gene expression in Roche LightCycler480. GAPDH expression and U6 expression were employed as internal control, respectively. The 2−∆∆Ct method was served for statistical analysis.
Primer sequences lists:

circ0093335‐Convergent‐F: ATGGACATACCTAATACTTTCCAGGAT.

circ0093335‐Convergent‐R: TCCAGGTAACGAACAATACACGT.

circ0093335‐Divergent‐F: ATGGACATACCTAATACTTTCCAGGAT.

circ0093335‐Divergent‐R: TCCAGGTAACGAACAATACACGT.

GAPDH‐F: ATTGTACAGCCCGTCCCCAA.

GAPDH‐R: GAGTCGGCTAGGTGCG.

miR‐338‐5p‐F: GTTCACCACCTTCTCCAC.

U6‐F: CTCGCTTCGGCAGCACA.

Total RNA from spinal cord tissues and cells was isolated using TRIzol reagent (TaKaRa, Dalian, China). Reverse transcription of miR-129-5p was synthesized using the miScript II RT kit (Invitrogen, Carlsbad, CA), and cDNA of mRNA was synthesized by using an iScript cDNA synthesis kit (Bio-Rad). For miR-129-5p, the qRT-PCR assays were carried out using an miRNA qPCR detection kit (Tiangen, Beijing). For mRNA, qRT-PCR was conducted by SYBR-Green Gene Expression Assay kit (Tiangen, Beijing) and performed on an ABI Prism 7900 HT (Applied Biosystems). The primers used were as follows: miR-129-5p F 5′-GGGGGCTTTTTGCGGTCTGG-3′, R: 5′-AGTGCGTGTCGTGGAGTC-3′; U6 F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, R 5′-CGCTTCAGAATTTGCGTGTCAT-3′; HBMG1 F 5′-AGGCTGACAAGGCTCGTTATG-3′, R 5′-TGTCATCCGCAGCAGTGTTG-3′; GAPDH F, 5′-GAAGATGGTGATGGGATTTC-3′, and R, 5′-AACGCTTCACGAATTTGCGT-3′. The relative expression of each gene was calculated using the 2^−∆∆C_t method [30 (link)].