Deoxyribonucleic acid of patients was obtained from peripheral blood using standard procedures. All of them were genotyped for vaspin rs2236242 (T/A) and rs35262691 (T/C) polymorphisms, previously associated with CV risk factors and/or reported as functional gene variants [21 (link), 22 (link)]), using pre-designed TaqMan probes (C___2786211_1_ and C___7854490_10, respectively). Additionally, the rs7159023 (G/A) polymorphism was assessed with a pre-designed TaqMan probe (C__29386750_10). This polymorphism is in complete linkage disequilibrium (r2 = 1) with rs77060950 (G/T), also previously associated with serum vaspin levels [23 (link)]. Genotyping was performed in a QuantStudio™ 7 Flex Real-Time polymerase chain reaction system, according to the conditions recommended by the manufacturer (Applied Biosystems, Foster City, CA, USA). Negative controls and duplicate samples were included to check the accuracy of the genotyping.
Quantstudio 7 flex real time polymerase chain reaction system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio™ 7 Flex Real-Time polymerase chain reaction system is a thermal cycler designed for real-time PCR analysis. It can perform precise quantification of nucleic acid targets.
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Lab products found in correlation
2 protocols using quantstudio 7 flex real time polymerase chain reaction system
1
Genotyping of Vaspin Polymorphisms
2
Quantitative Gene Expression Analysis of Colon Organoids
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The cells of colonic organoids, day 3 chips, and day 7 Colon Chips were harvested with RLT buffer from the RNeasy Mini Kit (74106; Qiagen, Hilden, Germany). RNA was extracted using the RNeasy Mini Kit (74106; Qiagen) followed by complementary DNA synthesis with the SuperScript VILO complementary DNA Synthesis Kit (1754250; Invitrogen). Reverse-transcription polymerase chain reaction was performed using TaqMan Fast Advanced Master Mix (4444965; Applied Biosystems, Foster City, CA), TaqMan Gene Expression Assays (Hs02786624_g1 for glyceraldehyde-3-phosphate dehydrogenase, Hs00358836_m1 for Kruppel-like factor 4, Hs00171942_m1 for SAM pointed domain ETS factor, Hs00356521_m1 for anterior gradient 2, protein disulfide isomerase family member, Hs00395669_m1 for resistin-like molecule β, Hs00175398_m1 for Fc-γ binding protein, Hs01086545_m1 for Kallikrein 1, Hs00983260_m1 for Meprin A subunit β, and Hs00976287_m1 for chloride channel accessory 1; Thermo Fisher Scientific), and run on a QuantStudio 7 Flex Real-Time polymerase chain reaction System (Applied Biosystems). All results were normalized relative to glyceraldehyde-3-phosphate dehydrogenase expression and day 0 organoid respective gene expression.
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