Superreal premix plus kit

Total RNA was extracted from rat nucleus pulposus cells or tissues by using the TRIzol RNA isolation protocol (Invitrogen). The cDNA was synthesized by using a FastKing RT kit (with gDNase) (Qiagen). RT-PCR and qRT-PCR were performed by using a SuperReal PreMix Plus kit (SYBR Green) (Qiagen). All primers were synthesized by Takara Bio, Inc. (Tokyo, Japan). IL-1β: F-CACCT CTCAA GCAGA GCACA G, R-GGGTT CCATG GTGAA GTCAA C; NOS2: F-GACCA GAAAC TGTCT CACCT G, R-CGAAC ATCGA ACGTC TCACA; IL-18: F-ATATC GACCG AACAG CCAAC, R-TTCCA TCCTT CACAG ATAGG G; GAPDH: F-GGCAC AGTCA AGGCT GAGAA TG, R-ATGGT GGTGA AGACG CCAGT A.

Total RNA was isolated from tissues using the RNA Pure Plant Kit (Qiagen, Germany). Purified RNA (about 2 μg) was reverse transcribed to synthetic cDNA using MMLV reverse transcriptase (Promega, USA). Quantitative reverse-transcription PCR (qRT-PCR) assays were performed with an ABI 7500 real-time PCR detection system (Applied Biosystems, USA) using the SuperReal PreMix Plus Kit (Qiagen, Germany). The reaction protocol was 40 cycles of 95 °C for 10 s (denaturation), 60 °C for 20 s (annealing), and 72 °C for 30 s (extension). The ΔΔ^Ct method was used to calculate the transcript levels of the relevant genes (Livak and Schmittgen 2001 (link)). The primers used to amplify the SOD gene were 5′-CGGTGCACCAGAAGACGAAG-3′ and 5′-GCCAGTCTTCCACCAGCATT-3′, and the product size was 198 base pair (bp). The CAT gene primers were 5′-TCCCAACTACCTGATGCTGC-3′ and 5′-GTTGGGCTTGCGTATGGTTG-3′, and the product size was 209 bp. The POD gene primers were 5′-TGGAACACAAGCACGAACCC-3′ and 5′-CCTTCCACAGCGTCTCGTT-3′, and the product size was 279 bp (Ramazan et al. 2021 (link)). The ZmTubulin1 (ID: Zm00001d013367) gene was used as an internal control. The primers used to amplify ZmTubulin1 were 5′-GTGTCCTGTCCACCCACTCTCT-3′ and 5′-GGAACTCGTTCACATCAACGTTC-3′, and the product size was 299 bp (Tian et al. 2019 (link)).

Total RNA was drawn from the tissue or neurons using a Trizol reagent (Trizol™ Reagent, Invitrogen). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed by using a SuperReal PreMix Plus Kit (SYBR Green) (Qiagen) on the Bio-Rad CFX96TM Real-Time System. GAPDH was amplified as an internal control. Primer sequences as follows (Liu et al., 2018 (link)): TLR4 F: 5’- TCACA ACTCG CCCAA GGAGG AA -3’, R: 5’- AAGAG ACCAC GGCAG AAGCT AG -3’; MyD88 F: 5’- CCACC TGTAA AGGCT TCTCG -3’, R: 5’-CTAGA GCTGC TGGCC TTGTT-3’; NLRP3 F: 5’- GCTAA GAAGG ACCAG CCAGA GT -3’, R: 5’- GAACC TGCTT CTCAC ATGTC GT -3’; GAPDH F: 5’- AACTT TGGCAT TGTGG AAGG -3’ R: 5’- GGATG CAGGG ATGAT GTTCT -3’.