A Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs, Small RNAs, and miRNAs Using a Non-Radiolabeled Approach.

Waqar Ahmad,Farah Mustafa,Jasmin Baby,Bushra Gull

doi:10.3390/cimb43020036

Waqar Ahmad, Farah Mustafa + Show 2 more

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https://doi.org/10.3390/cimb43020036

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Abstract

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 biotinylation, use of exonuclease, and bioimagers not reported earlier. We demonstrate that, while the NBs were sensitive for mRNAs and small RNAs, our LH protocol could efficiently detect these and miRNAs using less than 10–100 times the total amount of RNA, a sensitivity comparable to radiolabeled probes. Compared to NBs, LH was a faster, more sensitive, and specific approach for mRNA/small RNA/miRNA detection. A comparison of present work with six seminal studies is presented along with detailed protocols for easy reproducibility. Overall, our study provides effective platforms to study large and small RNAs in a sensitive, efficient, and cost-effective manner.

Highlights

Recent advances in RNA biology and techniques to study gene expression at the transcription level have witnessed unparalleled progress, with our understanding of different forms of RNAs being constantly challenged
Multiple protocols have been described for RNA extraction, RNA extracted through TRIzol is still preferable for molecular biology studies, especially when it comes to the isolation of all classes of RNAs, from the larger ribosomal and mRNAs in the kilobase range, to the small RNAs in the 100 to 200 nucleotide range, to the miRNAs in the 20–25 nt range
We developed non-radiolabeled, biotin-based-northern blots as well as highly sensitive liquid hybridization assays and systematically compared them for all three sizes of RNAs: mRNAs, small RNAs, and miRNAs

Summary

Introduction

Recent advances in RNA biology and techniques to study gene expression at the transcription level have witnessed unparalleled progress, with our understanding of different forms of RNAs being constantly challenged. The advent of comprehensive sequencing techniques has revealed that more than 80% of the total genome is extensively transcribed, with only a mere 1–2% of the transcripts coding for functional proteins [1] This highlights the regulatory nature of the remaining proportion of RNA molecules transcribed from “junk DNA” or regions that do not code for any protein. MiRNAs are synthesized through multiple enzymatic cleavage reaction by RNase-III-like enzymes, such as Drosha and Dicer [6,7,8,9], important for crucial cellular pathways, including cell development and differentiation [10,11], cell cycle regulation [12], and cell proliferation [13] Dysregulation of their expression leads to cell transformation and promotion of carcinogenesis [10,14,15,16,17]. These regulatory RNAs have been observed to function as both a friend and a foe of intracellular pathogens such as viruses [18,19,20,21,22]

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