Enhanced detection of small RNAs using a nonradioactive approach

Abstract

Recent advancements in high-throughput sequencing have led to the identification of many new classes of small noncoding RNAs such as endo-siRNAs. Unfortunately, reliable quantification of RNAs by sequencing is difficult due to artifacts arising from various factors involved in cDNA library preparation. Northern blot is one of the leading methods used to confirm the presence of a given RNA sequence because it can accurately quantify the cellular abundance, the size of the small RNA and reveal the presence of potential precursors and RNA isoforms. Here, we present a comprehensive description of LNA probe design along with a recently developed highly sensitive and cost-effective nonradioactive northern blot approach termed LED. LED combines a cross-linking method (EDC) and digoxigenin (DIG) labeling, and it can detect small RNAs with concentrations as low as 0.05 fmol and requires as little as a few seconds of membrane exposure for signal generation.