Quanti fast one step rt pcr kit

Total RNA was extracted using the Qiagen (GmbH, Hilden, Germany) RNAEasy Mini kit. The quantitative real-time RT-PCR was carried out for the analysis of host gene expression in muscles using gene-specific primers from Quanti Tect primer assay kit and Quanti Fast one-step RT-PCR kit (Qiagen, Germany). The thermal profile consists of 10 min of reverse transcription at 50°C one cycle and 5 min of polymerase activation at 95°C, followed by 40 cycles of PCR at 95°C for 10 s, 60°C for 30 s for combined annealing/extension. Following amplification, a melting curve analysis was performed to verify the authenticity of the amplified product by its specific melting temperature (Tm) with the melting curve analysis software of the Mx3005p. The threshold cycle (Ct) of gene of interest (GOI) and housekeeping gene (HKG) and the difference between their Ct values (Ct) were determined. Relative changes of gene expression were calculated using delta delta Ct method [31] (link) and the data were represented as fold up-regulation/down-regulation compared to the mock-infected group.

Articular hip and knee cartilage was snap‐frozen in liquid nitrogen and pulverized using a 6770 Freezer/mill (Spex Sample Prep). Total RNA was extracted from both powdered cartilage and primary chondrocytes using TRIzol and further purified using RNeasy columns. RIN values were >7, and 260:280 ratios were >1.7. Custom primers and FAM‐labeled probes were designed using Primer Express 3 software (Life Technologies) for qRT‐PCR. The qRT‐PCR was performed from 25 ng of total RNA in a one‐step reaction (QuantiFast One‐Step RT‐PCR kit; Qiagen) using a Roche LightCycler 480 II. The relative expression of lncRNAs was determined using the ΔΔC_t method, following normalization to 18S RNA. GAPDH expression (relative to 18S RNA) was comparable between non‐OA and OA cartilage in both hip and knee samples (see Supplementary Figure 1, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.39520/abstract).