Abi stepone real time polymerase chain reaction system

Urine sample collection, processing, and storage was performed in an aseptic manner by an experienced technician to avoid cross-contamination. Mid-stream urine samples were collected on the day of renal biopsy or at the time of visit for medical checkup and were centrifuged at 2,000 g for 20 min at room temperature. Cell pellets were separated on clean benches, subsequently transferred into RNA (Invitrogen, Carlsbad, CA), and stored at -80°C until required. All these processes were performed immediately after urine sample collection; therefore, the urine samples were stored within 1 hour of collection. Total RNA was extracted using the PureLinkTM RNA Mini Kit (Invitrogen), according to the manufacturer’s recommendations. The amount of total RNA (ug) was measured using a NanoDrop^® ND-2000 UV spectrophotometer (Thermo Scientific, Waltham, MA), cDNA synthesis was performed with the total RNA using M-MLV RT enzyme (200 U/µl; Mbiotech, Inc., Seoul, Korea), and the levels of gene expressions using each target primer and SYBR Green Master Mix (Applied Biosystems, Foster city, CA) were measured on ABI StepOne real-time polymerase chain reaction system (Applied Biosystems), as previously described (22 (link)). Each mRNA level was normalized by 18S rRNA used as an endogenous control for the 2-ΔΔCt method, and then log₁₀-transformed to reduce deviation.

The allelic discrimination of MUC6 polymorphisms rs61869016, rs6597947 and rs7481521 was assessed using an ABI StepOne real‐time polymerase chain reaction system (Applied Biosystems), SDS v3.0 software (Applied Biosystems) and the TaqMan assay.²⁴

Four AURKA SNPs, namely rs1047972 (C/T), rs2273535 (T/A), rs6024836 (A/G), and rs2064863 (T/G), were selected due to their considerable effects on other malignancies [16 (link),17 (link),18 (link)]. Regarding genotyping, DNA was first extracted from the leukocytes of venous blood samples from each participant using the QIAamp DNA kits (Qiagen, Valencia, Valencia, CA, USA) according to the manufacturer’s instructions. Subsequently, the allelic discrimination of the four AURKA SNPs was surveyed using the ABI StepOne Real-Time polymerase chain reaction (PCR) system (Applied Biosystems, Foster City, CA, USA). The findings of the real-time PCR were then analyzed using a Safety Data Sheet v3.0 (Applied Biosystems, Foster City, CA, USA) through the TaqMan assay technique to enhance PCR integrity.