16-cell of zebrafish development
Total RNA from embryos was isolated using Trizol reagent (Invitrogen). RNAs were fractioned on 15% denaturing polyacrylamide gels, and small RNAs were isolated and purified. Subsequently, small RNAs were ligated with both a 5’ adapter and 3’ adapter for reverse transcription using SuperscriptTM II reverse transcription kit (Invitrogen) following the manufacturer's instructions at 42 oC for 1 h and 70 oC for 15 min. After that, the reverse transcribed product, cDNA was amplified by the following PCR program: a 15-cycle reaction at 98 oC for 30 sec, followed by 15 cycles consisting of 10 sec at 98 oC, 15 sec at 72 oC, and then 10 min at 72 oC. After obtaining a 92bp DNA band on 6% denaturing PAGE gels, the PCR products were enriched by ethanol precipitation and purified using Spin-X filter columns (Fisher).
Huazhong University of Science and Technology
16-cell stage