18s ribosomal rna

RNA was extracted from frozen kidneys using TRIzol (Life Technologies, 15596-018, Waltham, MA) according to the manufacturer’s instructions. The RNA concentration was quantified using a NanoDrop 1000 Spectrophotometer. cDNA was generated using a PrimeScript RT Reagent Kit (TAKARA, RR037A, Shiga, Japan). The gene expression was quantified using a SYBR Green PCR kit using 10 ng of cDNA. The primers used for the quantification were designed by Hokkaido System Science Co., Ltd. (Sapporo, Japan). All experiments were performed in duplicate, and 18S ribosomal RNA (Qiagen) was utilized as an internal control. MiR was extracted using a miRNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. The cDNA was generated using a miScript II RT kit (Qiagen). The miScript SYBR Green PCR Kit (Qiagen) was used to quantify the miR expression using 3 ng of cDNA. The primers used to quantify Mm_miR-200b-1, Mm_miR-200b-3, Mm_miR-29-a, Mm_miR-29-b, Mm_miR-29-c, Mm_miR-34-a, Mm_miR-34-b and Mm_miR-34-c were included in the miScript primer assays (Qiagen). All experiments were performed in duplicate, and Hs_RNU6-2_1 (Qiagen) was utilized as an internal control (Supplementary Table).

PCR analysis was performed as described previously [41 (link)]. In brief, total RNA was isolated using the High Pure RNA Isolation Kit (Roche, Mannheim, Germany) and transcribed into cDNA using FastGene Scriptase II (NIPPON Genetics Europe; Düren, Germany) according to the manufacturer’s instructions. CD133 primers were 5′-TGGGGCTGCTGTTTATTATTCT-3′ and 5′- TGCCACAAAACCATAGAAGATG-3′ [42 (link)]. Primer assays (QuantiTect Primer Assay) for 18S ribosomal RNA were from Qiagen (Hilden, Germany). Amplification of cDNA was performed using PerfeCTa SYBR Green FastMix (Quanta Biosciences; Beverly, MA, USA) on a LightCycler 480 (Roche, Basel, Switzerland) instrument. Relative CD133 mRNA expression—normalized to 18S rRNA—was calculated by the ΔΔ cycle-threshold (Ct) method.