The steps of the experiments

1. Design of amiRNA Sequences

The target gene we want to silence is PGC-1a which is related to metabolism in several tissues. After inputting the sequence of PGC-1a to the Web MicroRNA Designer, the software will give us several potential amiRNA sequences which have high possibilities to silence the target genes. Although the sequences are picked out based on several principles, we still need to select the sequences by ourselves in the rules:

• It is preferable for all intended target genes to not have mismatches to the amiRNA at positions 2 to 12.
• AmiRNA candidates with one or two mismatches at the 3¢ end of the amiRNA (positions 18 to 21) should be preferred, since it has been suggested that perfectly matching amiRNAs might trigger so-called transitive siRNA formation, where amplification of sequences adjacent to the binding site is primed by the miRNA. These sequences could in turn themselves serve as silencing triggers and affect other, unintended genes.
• The absolute hybridization energy of the binding between amiRNA and the target sequence should be less than −30 kcal/ mole, and preferable be in the range between −35 and −40 kcal/ mole.
• The amiRNA binding site should be located within the coding region of the target gene, since UTRs are more likely to be misannotated. At least two amiRNAs per target gene or group of genes should be selected for experimental work. If several are selected, the amiRNAs should bind the target mRNA at different locations, since secondary structure is suspected to influence miRNA efficacy.