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. 2012 Sep 11;7(9):e44873. doi: 10.1371/journal.pone.0044873

Figure 7. Validation of miRNAs and their targets associated with mitochondria.

Figure 7

The enrichment of target mRNA of mitochondria-associated miRNAs with outer mitochondrial membrane were validated. (A) The association of 5S rRNA and negative control (hsa-miR-145) to mitochondria was analyzed. The relative enrichment of 5S rRNA and hsa-miR-145 was determined by qPCR as described in methods. (B) The miRNAs (frequency >1000) which were common in both the libraries were selected for validations by RT-qPCR. RNA was prepared from purified mitochondria and cDNA synthesized. The mean CT values of miRNAs (with CT <30 and distinct melt curve) from the mitochondria of both HEK293 and HeLa are listed. (C) The mitoplast was prepared from HEK293 as described in method section. The purity of mitoplast preparation was assessed by western blotting by probing with antibody against NDUFS2 (mitochondrial inner membrane protein) and cytochrome c (inter membrane space protein). TC: total cell; MP: mitoplast; MT: mitochondria. The targets of 3 miRNAs associated with mitochondria (let-7b: STAT3; hsa-miR-107: MFN2; hsa-miR-320a: XIAP) were determined by Starbase and validated by qPCR. (D) Validation of target mRNA associated with mitoplast as compared to mitochondria from HEK293. (E) Validation of target mRNA associated with RNase A treated mitochondria from HEK293.