Superscripttmii reverse transcription kit

Total RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Equal amounts (about 25 μg) of total RNA from four replicates of each time-point sample were mixed together for sRNA libraries construction. The sRNA libraries were constructed following the methods described by Lu et al [20 ]. Briefly, the total RNA was fractionated by 15% denaturing polyacrylamide gel (8 M urea) electrophoresis, and sRNAs in the range of 18–30 nt were excised and purified with a Spin-X cellulose acetate filter (2 mL, 0.45 um; Thermo Fisher, Waltham, MA, USA). After dephosphorylation and sequential ligation of 5ʹ- and 3ʹ- Solexa adaptors, the sRNAs were reverse-transcribed using superscript^TMII reverse transcription kit (Invitrogen), and then amplified by PCR with phusion High-Fidelity PCR kit (Finnzymes, Espoo, Finland) to produce sRNA sequencing libraries. Next generation sequencing was performed on an Illumina platform (Nextomics Science and Technology Limited, Wuhan, China). All the sequence data have been deposited as a series with the accession number GSE73657 at NCBI’ GEO database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE73657).

Zebrafish embryos were homogenised by addition of Trizol (Sigma, USA) and passed through a QIAshredder column (Qiagen, Netherlands). The RNA was then purified; First‐Strand cDNA Synthesis was performed using SuperScriptTM II Reverse Transcription kit (Invitrogen, UK) as per manufacturer's instructions. qPCR was performed using MESA GREEN qPCR MasterMix Plus for SYBR® (Eurogentec, UK) and an ABI 7900HT Sequence Detection System (Applied Biosystems, CA, USA). Quantification was carried out by fold expression change following irradiation (2^−ΔΔCt, relative to βactin and unirradiated control). Primer sequences are listed in Table 2.

Trizol reagent (Invitrogen) was used for all RNA extractions from cells and tissue samples from patients. To harvest miRNAs, 85% of ethanol was used to precipitate and wash RNA samples. LookOut® DNA Erase (Sigma-Aldrich) was used to digest all RNA samples to remove genomic DNAs. SuperScriptTM II reverse transcription kit (Invitrogen) was used to transcribe RNA samples into cDNAs, which were used as templates for performing qPCR assays using SYBR@ Premix Ex TaqTM (TaKaRa Bio Group). With GAPDH as endogenous control, the levels of CASC2 expression were measured. All PCR reactions were performed in triplicate and the 2^−ΔΔCT method was used to calculate the fold changes of gene expression.