A Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs, Small RNAs, and miRNAs Using a Non-Radiolabeled Approach.
Typhoon & Sapphire biomolecular imaging
biotinylation
exonuclease 1
liquid hybridization assay
mRNA
miRNA
non-radioactive
northern blotting
small RNA
Journal
Current issues in molecular biology
ISSN: 1467-3045
Titre abrégé: Curr Issues Mol Biol
Pays: Switzerland
ID NLM: 100931761
Informations de publication
Date de publication:
22 Jun 2021
22 Jun 2021
Historique:
received:
10
04
2021
revised:
07
06
2021
accepted:
16
06
2021
entrez:
2
7
2021
pubmed:
3
7
2021
medline:
28
12
2021
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 34206608
pii: cimb43020036
doi: 10.3390/cimb43020036
pmc: PMC8929067
doi:
Substances chimiques
DNA Probes
0
MicroRNAs
0
RNA, Messenger
0
Biotin
6SO6U10H04
Digoxigenin
NQ1SX9LNAU
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
457-484Subventions
Organisme : United Arab Emirates University
ID : 31R122
Organisme : United Arab Emirates University
ID : 31R140
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