Mx3005p real time qpcr system

The soleus muscles were used for this analysis. Total RNA was extracted from muscle samples using a RNeasy Fibrous Tissue Mini Kit (Qiagen, CA, USA). Total RNA was used as a template with a QuantiTect® Reverse Transcription Kit (Qiagen) to prepare cDNA, and real-time RT-PCR was performed using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies, CA, USA). The cDNA concentration of all samples was unified to 25 ng/μl, the cDNA was applied 0.2 μl to each well. The synthetic gene-specific primers are listed in Table 1. The threshold cycle (Ct) was determined using an Mx3005P Real-Time QPCR System (Agilent Technologies). The mRNA expression of target genes was calculated using the ΔΔct method.

The real-time fluorescence quantitative PCR (FQ-PCR) procedure was in accordance with previous reports [27 (link)]. It was performed with an Mx3005P real-time qPCR system (Agilent, California, USA) and Ultra SYBR Mixture (Cwbiotech, Beijing, China), using a 20-μl reaction solution containing 10 μl 2× Ultra SYBR Mixture (Cwbiotech, Bengjing, China), 0.5 μl of each primer (Table 1), 2 μl DNA template and 7 μl DNase/RNase-free water. The thermal cycler profile was 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 30 s. All samples were measured in triplicate. Differences in gene expression were calculated using the 2-cycle threshold method. β-actin was used as a reference gene. For analysis of mRNA expression of each validated gene, raw data were normalized against the values obtained for β-actin mRNA, and the fold changes in the expression of each gene in the infected group vs. the control group were determined. The standard curve for PCV2 was obtained using 10-fold dilutions of plasmid DNA for viral loads.

First-strand cDNAs were synthesized using a PrimeScript™ II 1st strand cDNA Synthesis Kit (Takara Bio Inc., Shiga, Japan). The resulting first-strand cDNAs were used as templates for qPCR. Real-time PCR was performed using Brilliant III Ultra-Fast SYBR^® Green QPCR Master Mix on an Mx3005p real-time QPCR system (Agilent Technologies). S. dulcis 18S rRNA gene (JF718778) was used for normalization. The sequences of primers used in this study are listed in Supplemental Table S7.

The soleus muscles were used for this analysis. Total RNA was extracted from muscle samples using a RNeasy Fibrous Tissue Mini Kit (Qiagen, CA, USA). Total RNA was used as a template with a QuantiTect® Reverse Transcription Kit (Qiagen) to prepare cDNA, and real-time RT-PCR was performed using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies, CA, USA). The cDNA concentration of all samples was unified to 25 ng/μl, the cDNA was applied 0.2 μl to each well. The synthetic gene-specific primers are listed in Table 1. The threshold cycle (Ct) was determined using an Mx3005P Real-Time QPCR System (Agilent Technologies). The mRNA expression of target genes was calculated using the ΔΔct method.

Total RNA was extracted from the infrapatellar fat pad and synovium samples (at At 7 wks; Sed + OA, n = 5; Ex + OA, n = 5; Sed + Sham, n = 5) with the use of an RNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia, CA). However, enough RNA could not be extracted from one sample in the Sed + Sham group and was therefore excluded from the analysis. Total RNA was used as a template with a QuantiTect Reverse Transcription Kit (Qiagen) to prepare cDNA, and a real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA). The cDNA concentration of all samples was unified to 25 ng/μL, and 0.2 μL of the cDNA was applied to each well. The synthetic gene-specific primers are listed below in Table 1. The cycle threshold (Ct) was determined using an Mx3005P Real-Time QPCR System (Agilent Technologies). The mRNA expression of target genes was calculated using the ΔΔct method. Regarding IL-1β mRNA, the samples obtained 7 days after the MIA or saline injections were used.