Based on the sequences of ORFs, primers for RT-qPCR were designed using Primer-Blast (www.ncbi.nlm.nih.gov/tools/primer-blast ) (Table 1 ). A. obscurus β-actin was used as a reference gene. The RT-qPCR was carried out using a Cycler iQ Real-Time PCR Detection System (Bio-Rad, Hercules) in a final volume of 20 μl reaction mixture containing 10 μl of UltraSYBR Mixture (CWBIO, Beijing, China), 0.5 μl of each primer, 1 μl of cDNA template, and 8 μl of ddH2O. The reactions were conducted as follows: 95°C for 10 min, 40 cycles of 15 s at 95°C, 1 min at 60°C. Each RT-qPCR reaction was performed in three biological replicates and each sample was technically repeated three times. The relative expression levels of Hsps were calculated by the 2−∆∆Ct method (Livak and Schmittgen 2001 (link)).
Cycler iq real time pcr detection system
Manufactured by Bio-Rad
Sourced in United States
The Cycler iQ Real-Time PCR Detection System is a laboratory instrument designed for the amplification and detection of nucleic acid sequences using real-time polymerase chain reaction (PCR) technology. The system provides advanced thermal cycling and optics capabilities to enable precise and sensitive quantification of target DNA or RNA in a sample.
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Lab products found in correlation
2 protocols using cycler iq real time pcr detection system
1
Quantifying Heat Shock Protein Expression
2
Quantitative Analysis of Collagen Genes
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Expression analysis of collagen-related genes was performed using a method developed by Livak et al. [16 (link)]. Total RNA was extracted from 100 mg muscle tissue using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommendations. Agarose gel electrophoresis (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the purity of total RNA, and RNA quality and quantity were analyzed according to their OD260/OD280 ratios. After isolation, the total RNA was reverse-transcribed by a cDNA reverse transcription kit (Vazyme, Nanjing, China). The expression level of the COL1A1, COL1A2, and COL3A1 genes was examined using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), with β-actin used as the reference gene (Table S1 ). Real-time quantitative polymerase chain reaction (qPCR) was performed in a Cycler I Q real-time PCR detection system (Bio-Rad, Hercules, CA, USA), and the relative expression of mRNA was calculated using the 2−ΔΔCT method.
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