The RNA from peripheral blood samples was extracted using the RNeasy Plus Universal Kit (Qiagen), and 1 μg of RNA was applied to synthesize cDNA by PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara Bio, Kusatsu, Japan). Template cDNAs were diluted 1:4, and the PON1 relative expression was quantified through the ABI 7500 Quantitative Real‐time PCR (qRT‐PCR) System (Applied Biosystems, Foster City, CA) and normalized with housekeeping gene GAPDH. The primers of qRT‐PCR amplification were designed using the software of Primer Premier 5, and their sequences are listed in Table S2 . After samples run in triplicate, we received the mean value. The relative quantitative method was implemented for the calculation of the level on PON1 mRNA.
Abi 7500 quantitative real time pcr qrt pcr system
Manufactured by Thermo Fisher Scientific
The ABI 7500 Quantitative Real-time PCR (qRT-PCR) System is a laboratory instrument designed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It is capable of precisely detecting and quantifying nucleic acid sequences in biological samples.
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2 protocols using abi 7500 quantitative real time pcr qrt pcr system
1
Quantification of PON1 Expression
2
Quantifying CREB5 mRNA Expression
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We used qRT‐PCR to validate the relative mRNA expression of the CREB5 gene corresponding to the loci above. The RNA of the samples was extracted with RNeasy Plus Universal Kit (Qiagen). The synthesis process of cDNA required the PrimeScript™ RT Reagent Kit and gDNA Eraser (TaKaRa Bio), and 1 μg RNA was applied. Template cDNAs were diluted 1:4. The relative expression of CREB5 gene was quantified with ABI 7500 Quantitative Real‐time PCR (qRT‐PCR) System (Applied Biosystems). The GAPDH gene was selected for normalization. The primers for qRT‐PCR amplification were designed with Primer Premier 5 (Table S2 ). After the samples were run in triplicate, the mean result was determined. The relative quantitative method was applied to calculate the mRNA levels of CREB5.
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