Fig. 4.

Effects of miR transfection on KRT RNA levels. Assessment with multiple KRT amplicons and Northern blot analyses. Transfections were performed as described and 48 h following transfection, RNA was purified. Panel A: QPCR analyses were performed for a second amplicon in KRT, KRT_1281–1381. The results were analogous to those obtained with the KRT amplicon KRT_548–648 (shown in Fig. 2). In addition to showing no change in KRT mRNA level, the ratios of the KRT_1281–1381/KRT_548–648 amplicons were constant at unity in all samples (Y-axis), suggesting that KRT mRNA was intact and that no preferential degradation had occurred. Panel B: Northern blot analyses were also performed (in triplicate), which also showed no change in mRNA levels (vs. GAPDH as control). In additional transfection experiments, protein was harvested and examined by immunoblot analyses as described. Three independent experiments were performed, and analyses were run in triplicate. No change in KRT protein levels was observed in the three experimental miR transfections with miRs targeting region 9 in KRT (lanes 8–10), and the other samples showed changes similar to those previously reported (shown in Fig. 2). Lanes in panels A and B show: (1) miR-let-miR-7c*; (2) miR-let-7f; (3) miR-let-7g; (4) miR-125b-1*; (5) miR-615-5p; (6) miR-138; (7) miR-let-7b; (8) miR-638; (9) miR-675; (10) miR-127-3p;and (11) transfection control. A random miR was also without effect (not shown).