Publisher Summary Antisense oligonucleotides (ASO) offer significant advantages over traditional methods in terms of specificity and versatility. ASOs validate the function of genes in vitro and in vivo. In addition, antisense oligonucleotides are developed as drugs with many products currently being tested in clinical trials. ASOs are short stretches of synthetic, chemically modified nucleic acids that use Watson–Crick base pairing to specifically hybridize to mRNA sequences. For optimal activity, oligonucleotides must first be taken up by the cell or the targeted organ, and the targeted mRNA sequence must then be accessible to the oligonucleotide. Small interfering RNA has been used successfully to inhibit gene expression in mammalian cells. Comparison of siRNAs to RNase H-dependent oligonucleotides showed that they exhibit similar degrees of inhibition of gene expression. It is ideal to have more than one active ASO against a given target when phenotypic end points are measured to insure specific effects. Some proteins with a long half-life time might require repeated transfections with the oligonucleotide before a decrease is observed.