Example of miRNA family inhibitor design. (A) miR-30 family: identification of a shared sequence unique for the miR-30 family (in bold). In this particular case it was possible to design one short, complementary inhibitor, miR-30 Family Inhibitor (FI), with high duplex melting temperature (Tm) that binds with high specificity to members of the miR-30 miRNA family. (B) miR-200 family: silencing of the miR-200 family cannot be achieved with a single inhibitor. However, the five family members can be addressed with three inhibitors. The miR-200 Family Inhibitor (FI)-2 is the result of an oligonucleotide synthesis with a mixed-base composition, giving rise to two oligonucleotides that differ at position 7 (from the 5’ end). M = A,C. Letters in color indicate positions in which the target sequence varies between family members.
(A) Sub-confluent hMADS cells were transfected with one of anti-miR control (anti-neg), anti-miR 30, pre-miR control (pre-miR-neg), pre-miR-30a, or pre-miR-30d and were induced to undergo adipocyte differentiation 3 days later. Differentiation was assessed at the indicated time points (D4 or D10) by photomicrographic recording (top row) and Oil red O plus crystal violet counterstaining (lower row). (B) Assessment of adipogenesis by GPDH enzymatic activity. Results are means of three culture wells (24-well plates). Error bars represent mean ± standard error of the mean (N = 3). *P < 0.05. (C) Expression of adipogenesis-induced genes (CEBPb, PPARg and FABP4) by qPCR. The level of expression of each gene in control cells (anti-miR-neg or pre-miRneg) was taken as 1. From Zaragosi et al. Genome Biology 2011, 12:R64. Reprinted with permission from BioMed Central.