2.8. Presence of Gene SRY in Renal Tissue

BMSCs and iPS were isolated from male rats and injected into female rats, enabling detection of the SRY gene (located on the Y chromosome ([GenBank: {"type":"entrez-nucleotide","attrs":{"text":"FJ168067.1","term_id":"208463432","term_text":"FJ168067.1"}}FJ168067.1])) as an index of engraftment. Total RNA was isolated from the kidney using a Trizol Reagent (Life Technologies), and RNA concentration was determined using Qubit fluorometer according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). Reverse transcriptase reactions were performed with a high-capacity RNA-to-cDNA kit (Applied Biosystems) using 1 μg of total RNA. qPCR was performed using the StepONE plus real-time PCR system (Applied Biosystems) and TaqMan probes (Applied Biosystems) for GAPDH (Rn99999916_s1; endogenous control gene) and SRY (Rn04224592_u1). The real-time PCR conditions consisted of an initial denaturation step of 10 minutes at 95°C, followed by 40 cycles at 95°C for 15 seconds, and at 58°C, for 1 minute.