Figure 4. Northern blot and Taqman QPCR analysis showing that novel candidate miRNAs with stem lengths of ≥30bp may comprise bona fide miRNAs.

(A) The expression of miRNA was examined by northern blotting using end-labeled oligonucleotide probes. A total of 10 candidate miRNAs were selected from Table 2 and their 500-nt precursors cloned in plasmids and overexpressed in 293T cells to detect processed 70-nt pre-miRNA and a mature miRNA. The numbers in parentheses represent the read frequency in MDA-MB-231 cells obtained from deep sequencing. The numbers at the bottom indicate the length of stem in individual candidate miRNAs. A synthetic ³²P-labeled RNA marker was used as a size marker. Asterisk indicate miRNAs selected for QPCR validation. Taqman miRNA assay showed expression of candidate miRNAs from each group by Ct values (B) and fold change (C). RNU48 was used as positive control and no-template control (NTC) used as negative control. (D) Box plot graph showing correlation between validated miRNA and non-validated miRNA with their stem length. Mann-Whitney test showing that the two groups are statistically different (p<0.001).