Api prism 7900ht sequence detection system

The levels of IL-17R mRNA in the dorsal horns of the spinal cord’s cervical and lumbar segments were identified. Total mRNA extraction was carried out in accordance with manufacturer’s instructions, and the RNA 4 Aqueous kit (Ambion Inc., Austin, TX, USA) was used for purification. Following the assessment of concentration, each mRNA sample underwent a reverse transcriptase reaction using a Retroscript kit from Ambion Inc. (Austin, TX, USA). The template for creating first-strand cDNA was total mRNA. The API Prism 7900HT Sequence Detection system was used to perform RT-qPCR with three independent repetitions in accordance with the SYBER Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) instructions. The reaction protocols consisted of 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C and 60 s at 60 °C. The normalized curve was used to calculate each gene’s expression. According to the delta-delta-Ct method, quantitative standardization was carried out in each sample using the expression of GAPDH (glyceraldehydes 3-phosphate dehydrogenase) as an endogenous control [27 (link)]. The data were shown as fold change relative to the control. The following forward and reverse primers were used: IL-17R (Forward: 5′-TACCACAGTTCCCAAGCC AGTT-3′; Reverse: 5′-GGGGAGTCAGGTCTGCTACG-3′), and GAPDH mRNA (5′-AGGTCGGTGTGAACGGATTTG-3′; 5′-TGTA GACCATGTAGTTGAGGTCA-3′).

Pentraxin-3 and nectin-1 gene expression levels in spinal cord dorsal horn, amygdala and cortex specimens were assessed. The total mRNA was extracted, as described by the manufacturer of the RNA 4 Aqueous kit (Ambion, Austin, TX, USA). The Retroscript kit (Ambion) was employed for reverse transcription of a total of 1 mg total mRNA. qRT-PCR was carried out on an API Prism 7900HT Sequence Detection system with the SYBR Green PCR Master Mix kit (Applied Biosystems, Foster city, CA, USA), as directed by the manufacturer. Amplification was performed at 50 °C (2 min) and 95 °C (10 min), followed by 40 cycles at 95 °C (15 s) and 60 °C (60 s). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was utilized for normalization in the 2^−∆∆Ct method [26 (link)]. Primers were: pentraxin-3, sense 5′-CCTGCTTTGTGCTCTCTGGT-3′ and antisense 5′-TCTCCAGCATGATGAACAGC-3′; nectin-1, sense 5′-CCGTAAAGGTCAAGGGCAGAG-3′ and antisense 5′-GTGCCTGTCCCTTGTCCA-3′; GAPDH, sense 5′-AACAGCAACTCCCACTCTTC-3′ and antisense 5′-CCTCTCTTGCTCAGTGTCCT-3′.