Urine single spot samples were collected at the day of biopsy and stored at −80 °C until miRNA extraction. MagMAX mirVana Total RNA Isolation Kit (Applied Biosystems, Thermo Scientific, Vilnius, Lithuania) with MagMAX magnetic bead technology was used to extract miRNA from urine samples according to the manufacturer’s protocol. The extracted miRNA was reverse transcribed into complementary DNA (cDNA) using the TaqMan Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, Thermo Scientific, Vilnius, Lithuania).
Real-time quantitative polymerase chain reaction (qPCR) was performed to quantify miR-21 and microRNA-191-5p (miR-191) using TaqMan Advanced miRNA Assay and TaqMan Fast Advanced Master Mix (Applied Biosystems, Thermo Scientific, Vilnius, Lithuania). TaqMan microRNA assays IDs were 477975_mir (hsa-miR-21-5p) and 477952_mir (hsa-miR-191-5p).
MiR-191 was used for normalization as it is described as a stable gene showing a low degree of variation and high stability value [27 (link),28 (link)]. The relative gene expression levels were expressed by the ΔCt values (ΔCt = CtmiR-21 − CtmiR191, where Ct is the cycle threshold value) [29 (link)]. Lower ΔCt values indicate a higher relative expression of the miR-21. All experiments were carried out in duplicate and the arithmetic mean value was used for analysis.
Real-time quantitative polymerase chain reaction (qPCR) was performed to quantify miR-21 and microRNA-191-5p (miR-191) using TaqMan Advanced miRNA Assay and TaqMan Fast Advanced Master Mix (Applied Biosystems, Thermo Scientific, Vilnius, Lithuania). TaqMan microRNA assays IDs were 477975_mir (hsa-miR-21-5p) and 477952_mir (hsa-miR-191-5p).
MiR-191 was used for normalization as it is described as a stable gene showing a low degree of variation and high stability value [27 (link),28 (link)]. The relative gene expression levels were expressed by the ΔCt values (ΔCt = CtmiR-21 − CtmiR191, where Ct is the cycle threshold value) [29 (link)]. Lower ΔCt values indicate a higher relative expression of the miR-21. All experiments were carried out in duplicate and the arithmetic mean value was used for analysis.
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