biostudies-literatureAhmad WUnited Arab Emirates University457-484https://www.ebi.ac.uk/biostudies/studies/S-EPMC8929067biostudies-literatureUnknown43(2)Northern blotting (NB), a gold standard for RNA detection, has lost its charm due to its hands-on nature, need for good quality RNA, and radioactivity. With the emergence of the field of microRNAs (miRNAs), the necessity for sensitive and quantitative NBs has again emerged. Here, we developed highly sensitive yet non-radiolabeled, fast, economical NB, and liquid hybridization (LH) assays without radioactivity or specialized reagents like locked nucleic acid (LNA)- or digoxigenin-labeled probes for mRNAs/small RNAs, especially miRNAs using biotinylated probes. An improvised means of hybridizing oligo probes along with efficient transfer, cross-linking, and signal enhancement techniques was employed. Important caveats of each assay were elaborated upon, especially issues related to probe bioCurrent issues in molecular biologyA Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs, Small RNAs, and miRNAs Using a Non-Radiolabeled Approach.PMC892906731R12231R140Baby JAhmad WGull BMustafa FfalseA Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs, Small RNAs, and miRNAs Using a Non-Radiolabeled Approach.Northern blotting (NB), a gold standard for RNA detection, has lost its charm due to its hands-on nature, need for good quality RNA, and radioactivity. With the emergence of the field of microRNAs (miRNAs), the necessity for sensitive and quantitative NBs has again emerged. Here, we developed highly sensitive yet non-radiolabeled, fast, economical NB, and liquid hybridization (LH) assays without radioactivity or specialized reagents like locked nucleic acid (LNA)- or digoxigenin-labeled probes for mRNAs/small RNAs, especially miRNAs using biotinylated probes. An improvised means of hybridizing oligo probes along with efficient transfer, cross-linking, and signal enhancement techniques was employed. Important caveats of each assay were elaborated upon, especially issues related to probe bio2021-01-01T00:00:00Z2021 Jun2025-04-04T09:41:06.029Z2025-04-04T09:41:06.029ZS-EPMC89290673420660810.3390/cimb43020036