Lightcycle 480 2

To evaluate the accuracy of data from HiSeq, qRT-PCR (Wang et al., 2011) was used to quantitatively detect several miRNAs differentially expressed among the sham group, IRI group, and apelin-13 group. In brief, RNAs were isolated and cDNA was reverse-transcribed using an NCode™ VILO™ miRNA cDNA Synthesis Kit (Invitrogen). The PCR amplification was performed in LightCycle 480II (Roche, Basel, Switzerland) with the NCode™ EXPRESS SYBR GreenER™ Kit (Invitrogen). The reaction procedure was performed as follows: 2 minutes at 50°C, 2 minutes at 95°C, and then 40 cycles of denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 60 seconds. The nuclear RNA U6 was used as a loading control. All reactions were carried out in triplicate. The relative expression level of miRNA was calculated and normalized using the 2^−ΔΔCT method.

Total RNA was extracted from all tissues by trizol reagent (Code No 9108, Takara Bio Inc., Shiga, Japan) according to the manufacturer’s protocol. The quality and quantity of extracted RNA was measured by a NanoDrop Spectrophotometer (Shimadzu Biotech, Beijing China). The extracted RNA was determined to be pure only when the A260/A280 ratio is 1.8 to 2.1. For the reverse transcription reaction, 1 µg of total RNA was used by PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) (Code No RR047A, Takara Bio Inc., Kusatsu, Japan). The qRT-PCR was conducted by Light Cycle@480 II (Roche, Basel, Switzerland) by using a SYBR^® Premix DimerEraser™ (Perfect Real Time) (Code No: RR091A, Takara Bio Inc.) in a 20 µL mixture according to the manufacturer’s protocol. The reaction was carried out at 95°C for 30 s for one cycle, and 95°C for 5 s, 55°C for 30 s, and 72°C for 30 s for 45 cycles. Primers used in these reactions are listed in Table 1. The relative expression of target gene was normalized to the endogenous gene β-actin, calculated by the 2^−ΔΔCt method. All of this qRT-PCR was conducted in duplicate.