Quantitect quantiscript reverse transcriptase and rt primer mix

For reverse-transcription polymerase chain reaction (RT-PCR), total RNA was isolated using TRIzol reagent (Invitrogen). RNA concentration was measured by NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and cDNA was synthesized from a total of 1 μg RNA using QuantiTect^® Quantiscript reverse-transcriptase and RT Primer Mix (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. RT-PCR was performed using Taq PCR Master Mix Kit (Qiagen), according to standard protocols. The sequence of beta actin (ACTB) and cathepsin S (CTSS) gene-specific primers were as follows: ACTB (5′-CATGTACGTTGCTATCCAGGC-3′(sense) and 5′- CTCCTTAATGTCACGCACGAT-3′(antisense); product size 250bp) and CTSS (5′- TGACAACGGCTTTCCAGTACA-3′(sense), 5′- GGCAGCACGATATTTTGAGTCAT-3′(antisense)]; product size 113 bp). PCR products were analyzed with 1% agarose gel electrophoresis.

For the detection of IL-1β and PAPP-A mRNA expression, total RNA in the harvested cells (1x10⁶ cells) was homogenized in triazole. The homogenate was centrifuged for 1 min (13,000×g, 4°C). The RNA concentration was measured by NanoDrop ND-2000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE), and cDNA was synthesized using a total of 1 μg RNA using QuantiTect® Quantiscript reverse-transcriptase and RT Primer Mix (Qiagen), according to the manufacturer’s protocol. cDNA was synthesized by reverse transcription, and then q-PCR amplification was performed using it as a template. The reaction procedure was: denaturation at 95°C for 5 s, annealing at 56°C for 10 s, and extension at 72°C for 25 s, for a total of 40 cycles. IL-1β, PAPP-A, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected in the cells. After the reaction, the respective amplification and melting curves were analyzed. PCR primers (Table 1) were synthesized by Wuhan Tianyi Huayu Gene Technology Co., Ltd.