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. 2016 Oct 13;6:35026. doi: 10.1038/srep35026

Figure 2. Analysis of 47S pre-rRNA level.

Figure 2

(a) K562C cells were incubated with vehicle (−dox) or with doxycycline for 4 days (+dox) whereas K562, 22RV1 and HEK293 cells were transfected with unrelated siRNA (siCNT) or siRNA specific for RPS19 (siS19), RPS6 (siS6) or RPL11 (siL11). Total RNA was analyzed by RT-qPCR with primers specific for 5′ETS region of 47S rRNA and β-actin. The results of triplicate RT-qPCR from three independent RNA preparations are reported as a column plot of the mean ± s.e.m. of 47S rRNA/actin mRNA (siS19 is from a single triplicate experiment). (b) Total RNA extracted from K562S cells incubated with doxycycline for 4 days to induce a scrambled siRNA (+dox), from K562C or from TF-1C cells, untreated (−dox) or incubated with doxycycline for 4 days to induce an siRNA against the S19 mRNA (+dox) was analyzed by Northern blot with a 5′ETS oligonucleotide probe (indicated in Fig. 1b). An example of Northern blot is shown on the left. Uncropped original images are shown in Supplementary information. The results from three independent RNA preparations are reported as a column plot of the mean ± s.e.m. of 47S rRNA/actin mRNA. (c) HEK293 cells were transfected with control (siCNT) or RPS19 specific siRNA (siS19). Part of siS19 cells were then transfected with an RPS19 overexpressing plasmid (siS19 + S19). RNA was analyzed by RT-qPCR with primers specific for 5′ETS region of 47S rRNA and β-actin. The results of triplicate qPCR from three Reverse Transcription reactions are reported as a column plot of the mean ± s.e.m. of 47S rRNA/actin mRNA. (d) RNA isolated from cells treated as described in panel a, was analyzed by RT-qPCR with primers specific for RPS19, RPS6, RPL11 and β-actin. The results of triplicate RT-qPCR from three independent RNA preparations are reported as a column plot of the mean ± s.e.m. of RP mRNA/actin mRNA.