Identification and characterization of the Chinese giant salamander (Andrias davidianus) miRNAs by deep sequencing and their targets predication
Total RNA was extracted from animal tissues using TRIzol reagent (Invitrogen, USA) and then was treated with RNase-free DNase I (Takara, China) to remove any contaminating genomic DNA according to the manufacturer's protocol. The quantity and purity of total RNA was monitored via analysis by NanoDrop ND-1000 spectrophotometer (Nano Drop, USA) at 260/280 nm (ratio=2.0). The integrity of total RNA was tested via analysis by Bioanalyzer 2100 and RNA 6000Nano LabChip Kit (Agilent, USA) with RIN number>7.0. Briefly, small RNAs of 18-35 nt in length were first isolated from the total RNA by size fractionation. Then, these small RNAs were ligated with 5'-RNA and 3'-RNA adapters and subsequently reverse transcription PCR was used to create cDNAs. The amplified cDNAs were purified and sequenced with Illumina Hiseq 2500 platform.