LncRNA Knockdown Using Gapmer Antisense Oligonucleotides.

Long non-coding RNAs (lncRNAs) are a class of RNA that have diverse intracellular regulatory and structural roles. Because of their wide assortment of functions, lncRNAs have varied subcellular distributions and expression levels in the nucleus and/or cytoplasm of a cell. Even though nearly 100,000 human lncRNAs have been annotated in LncBook 2.0, only a small fraction have empirically validated functions. RNA knockdown, using antisense oligonucleotides (ASOs) or small interfering RNAs (siRNAs), is a relatively commonplace laboratory strategy used to functionally characterize an RNA. These RNA knockdown technologies can also have therapeutic benefits to treat a wide variety of genetic or infectious diseases, as evidenced by the several ASO and siRNA drugs that are currently FDA-approved or in clinical trials. This protocol describes the use of validated gapmer ASOs to knock down human MALAT1, a nuclear-retained lncRNA that is upregulated in multiple cancer cells. Methods used include cationic lipid transfection into HeLa cells, RNA isolation, and RT-qPCR analysis of the RNA knockdown levels.