Mir 335

Experiments were performed on kidneys harvested on D2. We extracted miRs using an isolation kit (mirVana; Thermo Fisher Scientific), and we used total RNA enriched with miRs. For reverse-transcriptase reaction, 5 ng of RNA was used, employing the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific). The miRs studied were miR-29a, miR-29b, miR-335 and miR-34a (Applied Biosystems; Thermo Fisher Scientific). RNU48, RNU44, U47 and U6 (Applied Biosystems; Thermo Fisher Scientific) were tested as possible housekeeping genes; we found U6 to be the best suited and used it in our analysis.
To perform a quantitative polymerase chain reaction (qPCR), we used TaqMan Universal PCR Master Mix II (Thermo Fisher Scientific). The data were analysed with DataAssist software (Applied Biosystems; Thermo Fisher Scientific) and relative gene expression calculated as 2^–ΔΔCt, where Ct is the threshold cycle.

Total RNA was isolated from skeletal muscles that were treated in 0.2% Collagenase Type 2 (Worthington) using miRNeasy Micro Kit (QIAGEN) according to the manufacture’s instructions. For analyses of mRNA expression, cDNA synthesis with oligo dT primers (Invitrogen) was performed using SuperScript III Reverse Transcriptase (Invitrogen). For analyses of miR-335 expression, reverse transcription was performed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacture’s instructions. qRT-PCR was performed using Power SYBR Green PCR Master Mix for mRNAs or TaqMan Universal PCR Master Mix (Applied Biosystems) for miRNAs on StepOnePlus (Applied Biosystems). Sequences of the primers are listed in S2 Table [5 (link),31 (link)–33 (link)], snoRNA-202 (Assay ID: 001232, Applied Biosystems) and miR-335 (Assay ID: 000546, Applied Biosystems).