Biotin based Northern Blotting (BiNoB): A Robust and Efficient Alternative

Abstract

Advancements in sequencing technology have led to the emergence 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 their regulatory roles and disease mechanisms. Northern blotting, which used to be considered the gold standard for differential expression analysis, poses challenges due to various limitations associated with RNA quality, integrity, radioactivity, reagents, and the expenses associated with it. In this study, we employed a biotin-based Northern blotting approach, BiNoB, which offers advantages over traditional methods. In this study, we comprehensively targeted various RNA types, making this technique a versatile tool for RNA detection. Additionally, we conducted a comparison between 3-end labeled probes, which were labeled in-house, and 5-end labeled probes, which were commercially obtained. Remarkably, the results revealed significantly increased sensitivity with 3-end labeled probes. Furthermore, we prepared an in-house buffer and compared its performance with the commercially available ULTRAhyb buffer, which exhibited comparable sensitivity, indicating that the in-house buffer is a cost-effective alternative. Intriguingly, our findings showed that as little as 1{micro}g of total RNA proved to be adequate for the detection of small RNAs like tRNAs and their derived fragments.