Sybr green fluorescent dye

Total RNA was extracted by miRNA isolation kit, and the RNA was removed by DNase I (both from Ambion, Austin, TX, USA) to test concentration, purity and integrity. RNA was reverse transcribed to cDNA by Reverse Transcription kit (Qiagen GmbH, Hilden, Germany). SYBR-Green Fluorescent dye (Sigma-Aldrich) was used for qPCR, performed on LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) apparatus. Primers were designed according to the GenBank website, with internal reference for company, and the RNA was removed using DNase I (Ambion) Technology Co., Ltd. PCR reaction conditions were: Pre-denaturation at 94°C for 5 min, 94°C for 30 sec, 63°C for 30 sec, 72°C for 30 sec, a total of 35 cycles, with overall elongation for 7 min at 72°C. The results were expressed by 2^−ΔΔCq method.

RNA obtained from different affinity-purified ribosomal particles or sucrose gradient fractions was extracted from the eluates using phenol–chloroform followed by overnight precipitation with 70% ethanol. Alternatively, the RNA Clean & Concentrator kit from Zymo Research has been used for RNA extraction. For detection of various snoRNAs, RNA samples were separated on 6% polyacrylamide/6 M urea gels for 3 h at 400 V, followed by 40 min Sybr Green fluorescent dye staining according to the manuals (Sigma-Aldrich, S9305). For northern blot analysis, the RNA was transferred to a nylon membrane (GE Healthcare) and crosslinked at 254 nm for 2 min. The oligonucleotide probes for Northern blot analysis which have been labelled with γ-³²P-ATP are listed in Table 3.