Total RNA was isolated from TRIzol by chloroform/ethanol extraction, precipitated, transcribed, and reverse-transcribed into cDNA, according to the manufacturer’s instructions. qRT-PCR was performed using a Bio-Rad CFX-96 touch system (United States) and iCycler technology. qRT-PCR primers were designed by Sangon Biotech, as shown in Table 1 . Amplification was performed according to the instructions of the TB Green kit (TaKaRa, Dalian, China), and the data were analyzed by LightCycler® 96 SW 1.1.
Lightcycler 96 sw 1
Manufactured by Takara Bio
Sourced in China
The LightCycler® 96 SW 1.1 is a real-time PCR system designed for quantitative and qualitative nucleic acid analysis. It features a 96-well format and a high-performance optical system for accurate and reliable results.
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Lab products found in correlation
2 protocols using lightcycler 96 sw 1
1
Quantitative Real-Time PCR Analysis of Gene Expression
2
QS-Related Genes in P. aeruginosa Biofilm
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Genes related to QS-mediated bio lm formation, such as the AHL receptor protein genes lasR, pqsA, and cdrA associated with bio lm formation, were retrieved from GenBank, and the rpsl gene was selected as the internal control for normalizing gene expression (Yin et al. 2021 (link)). The PvdQ enzyme (0.1 mg/mL) was grown overnight, at 28°C and 150 rpm, in LB medium containing P. aeruginosa, with three parallel settings for each group. Total RNA was extracted from bacterial somatic cells using the Total RNA Extraction Kit. The mass concentration of RNA was then analyzed by a Ultramicro spectrophotometer (TIANGEN, Beijing, China). The cDNA was generated from the total RNA using the PrimeScript TM -RT-reagent-Kit (TaKaRa, Dalian, China) and used as a template for the qRT-PCR (LightCycler® 96 SW 1.1). The mRNA expression levels of the QS-related genes were measured using the LightCycler® real-time PCR system and the TB Green® Fast qPCR Mix (2 Conc.) kit (TaKaRa, Dalian, China).
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