Trizol total rna reagent

Total RNA was extracted using the Trizol total RNA reagent (TIANGEN, China) and reverse-transcribed into cDNA using the FastKing RT Kit (TIANGEN, China). qRT-PCR was performed using the SuperReal PreMx Plus reagent (TIANGEN, China), with the following primers (GENERAL BIOL, China): HIPK3 (NM_031144.3; 137 bp), forward: 5′-TCACAGAGGCTTGGAGACTG-3′ and reverse: 5′-ACAACATGTGCGATGCCTAC-3′; beta-actin (NM_031787.2; 173 bp), forward: 5′-CACCATGTACCCAGGCATTG-3′ and reverse: 5′-CCTGCTTGCTGATCCACATC-3′. Reaction conditions were as follows: pre-denaturation at 95 °C for 15 min, followed by 40 cycles of denaturation at 95 °C for 10 s, and annealing and extension at 60°C for 32 s. Beta-actin served as an internal control. The miRcute miRNA Isolation Kit was used to extract miRNA and cDNA was generated with miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN, China). qRT‐PCR was carried out using the miRcute Plus miRNA qPCR Kit (TIANGEN, China) using the following reaction conditions: pre-denaturation at 95°C for 15 min, followed by 45 cycles of denaturation at 94°C for 20 s, and annealing and extension at 60°C for 34 s. Data were normalized to U6 spliceosomal RNA. The upstream and downstream miR-21 and U6 primers were designed by RiboBio Corporation (China).

The rats in the 4 groups were anesthetized with 10% chloral hydrate (3 mL/kg, i.p.). DRGs were isolated immediately and flushed with ice-cold PBS. Total RNA samples were prepared using TRIzol Total RNA Reagent (Beijing Tiangen Biotech Co.). The cDNA synthesis reaction was performed with 2 μg of total RNA and the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas, Burlington, Ontario, Canada). Primers were designed with Primer Express 3.0 software (Applied Biosystems), and the sequences were as follows: Quantitative real-time PCR (qPCR) was performed using the SYBR® Green MasterMix in an ABI PRISM® 7500 Sequence Detection System (Applied Biosystems, Inc., Foster City, CA). Gene expression was quantified using the ΔΔCT method with CT as the threshold cycle. The relative levels of target genes, which were normalized to the sample with the lowest CT, were reported as 2^−ΔΔCT.

The rats in all groups were anesthetized by 10% chloral hydrate (3 ml/kg, i.p.) on the 14th day after the operation. The L4–6 DRGs were immediately isolated and flushed with ice-cold phosphate-buffered saline (PBS). The total RNA samples were prepared from the L4–L6 DRGs of each group using the TRIzol Total RNA Reagent (Beijing Tiangen Biotech Co.). The cDNA synthesis was performed with 2 μg of total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, United States). The primers were designed with Primer Express 3.0 software (Applied Biosystems, Inc., Foster City, CA, United States) and the following sequences: P2X7, forward 5′-GAGTCCGAGGCAATCTAATG-3′; reverse 5′-CTGTGATCCCAACAAAGGTC-3′; and β-actin, forward 5′-TAAAGACCTCTATGCCAACACAGT-3′, reverse 5′-CACGATGGAGGGGCCGGACTCATC-3′. Quantitative PCR was performed using the SYBR^® Green Master Mix in an ABI PRISM^® 7500 Sequence Detection System (Applied Biosystems, Inc., Foster City, CA, United States). The quantification of gene expression was performed using the ΔΔCT calculation with CT as the threshold cycle. The relative levels of target genes, normalized to the sample with the lowest CT, were given as 2^-ΔΔCT (Tu et al., 2013 (link)). The β-actin was used to be internal control in the groups. The relative expression levels of mRNA in the all groups were normalized to β-actin.