دورية أكاديمية
A Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs, Small RNAs, and miRNAs Using a Non-Radiolabeled Approach
| العنوان: | A Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs, Small RNAs, and miRNAs Using a Non-Radiolabeled Approach |
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| المؤلفون: | Waqar Ahmad, Bushra Gull, Jasmin Baby, Farah Mustafa |
| المصدر: | Current Issues in Molecular Biology, Vol 43, Iss 2, Pp 457-484 (2021) |
| بيانات النشر: | MDPI AG, 2021. |
| سنة النشر: | 2021 |
| المجموعة: | LCC:Biology (General) |
| مصطلحات موضوعية: | northern blotting, liquid hybridization assay, exonuclease 1, non-radioactive, biotinylation, mRNA, Biology (General), QH301-705.5 |
| الوصف: | 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. |
| نوع الوثيقة: | article |
| وصف الملف: | electronic resource |
| اللغة: | English |
| تدمد: | 1467-3045 1467-3037 |
| العلاقة: | https://www.mdpi.com/1467-3045/43/2/36Test; https://doaj.org/toc/1467-3037Test; https://doaj.org/toc/1467-3045Test |
| DOI: | 10.3390/cimb43020036 |
| الوصول الحر: | https://doaj.org/article/87bc01d4e4e246dcb9e77b973b214008Test |
| رقم الانضمام: | edsdoj.87bc01d4e4e246dcb9e77b973b214008 |
| قاعدة البيانات: | Directory of Open Access Journals |
Full Text Finder | تدمد: | 14673045 14673037 |
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| DOI: | 10.3390/cimb43020036 |