Blood samples were collected from all subjects and genomic DNA was extracted from the buffy coats of centrifuged blood using the Puregene DNA purification kit (Gentra Systems, Inc., Minneapolis, MN, USA). The database single-nucleotide polymorphism identification numbers for the ADD1 (Gly460Trp), AGT (Gly6Ala), AT1 (Ala1166Cys), and GNB3 (Cys825Thr) polymorphisms were rs4961, rs5051, rs5186, and rs6489738, respectively. The TaqMan method was used for genotyping. Probes and primer mixtures were selected from the commercial database of the TaqMan probe system (myScience; Applied Biosystems, Foster City, CA, USA).25 (link) Genomic DNA (5 ng) was added to the wells of a 384-well plate and air-dried for use as templates for the reaction. The polymerase chain reaction mixture contained 2.5 µL of probe mix and the same amount of Master Mix solution (Applied Biosystems). The standard thermocycle parameters provided by the manufacturer were used. An ABI 7900HT apparatus (Applied Biosystems) was used for reaction, genotype calling, and data exporting. The ADD1 T allele, AGT A allele, AT1 C allele, and GNB3 T allele were previously identified as candidate gene polymorphisms responsible for salt-sensitive HT.17 (link),18 (link),26 (link)
Taqman probe system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TaqMan probe system is a real-time PCR (polymerase chain reaction) technology used for the detection and quantification of specific DNA or RNA sequences. It utilizes a fluorescent probe that binds to the target sequence, allowing for the continuous monitoring of the amplification process. The system provides a sensitive and accurate method for analyzing gene expression, detecting pathogens, and performing other nucleic acid-based analyses.
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3 protocols using taqman probe system
1
Genotyping of Hypertension-associated Polymorphisms
2
Quantifying Stem Cell Gene Expression
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Total RNA was isolated by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Quantitative reverse transcriptase polymerase chain reactions (qRT-PCR) were performed by the Taqman probe system from Applied Bio Systems (Foster City, CA) by using the following products RhoC:Hs00733980_m1, Sox2:Hs01053049_s1, Nanog: Hs02387400_g1 and Oct3/4 (POUF1) Hs01895061_u1. OZ1 and G3PDH were used as the data normalizers. Relative changes in gene expressions were calculated using the 2−(−Δ)CT method [24] (link).
3
Quantitative Analysis of RhoC and miR-138
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