Biotin‐Based Northern Blotting (BiNoB): A Cost‐Efficient Alternative for Detection of Small RNAs

Abstract Advances in sequencing technology have led to the discovery of diverse types of regulatory RNAs. Differential transcript levels regulate cellular processes and influence disease severity. Identifying these variations through reliable methods is crucial for understanding the regulatory roles and disease mechanisms of regulatory RNAs. Northern blotting, which is considered the gold standard for differential expression analysis, poses challenges due to various limitations associated with RNA quality and integrity, radioactivity exposure, and associated reagents and expenses. In this protocol, we employ a biotin‐based northern blotting (BiNoB) approach that is both convenient and inexpensive, eliminating the need for specialized settings as required with radioactivity‐based northern blotting. We comprehensively target various RNA types, making this technique a versatile tool for RNA detection. Additionally, we conduct a comparison between 3′‐end labeled probes that were labeled in‐house and 5′‐end labeled probes that were obtained commercially. Remarkably, our results reveal relatively higher sensitivity with 3′‐end labeled probes. Furthermore, we demonstrated that the use of an in‐house buffer offered comparable sensitivity to a commercially available buffer, providing another cost‐effective alternative. We also aimed to determine the minimum quantity of total RNA required to detect small non‐coding RNAs such as tRNA fragments. Whereas previous studies reported the use of 5‐10 µg total RNA for tRNA fragment detection, our findings revealed that as little as 1 µg total RNA is sufficient to detect small RNAs like tRNAs and their fragments. This concentration may vary depending on the expression levels of the specific RNAs being detected. © 2024 Wiley Periodicals LLC. Basic Protocol : Biotin‐based northern blotting