Evaluating the knockdown activity of MALAT1 ENA gapmers in vitro

Abstract

Antisense oligonucleotides (ASOs) are widely used for the identification of gene functions and regulation of genes involved in different diseases for therapeutic purposes. For in vitro evaluation of the knockdown activity of gapmer ASOs, we often use lipofection or electroporation to deliver gapmer ASOs into the cells. Here, we describe a method for evaluating the knockdown activity of gapmer ASOs by a cell-free uptake mechanism, termed as gymnosis, using MALAT1 gapmer ASOs modified with 2′-O-methoxyethyl RNA (2′-MOE) or 2′-O,4′-C-ethylene–bridged nucleic acid (ENA). This method is robust because it does not involve the use of any transfection reagent and has minimal effects on cell growth. Further, we describe a convenient technique for performing one-step reverse transcription and real-time qPCR using cell lysates without RNA extraction. Data for up to 96 samples can be obtained following these methods.