Academic Journal
Streamlined Full-Length Total RNA Sequencing of Paraformaldehyde-Fixed Brain Tissues
| العنوان: | Streamlined Full-Length Total RNA Sequencing of Paraformaldehyde-Fixed Brain Tissues |
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| المؤلفون: | Bingqing Ji, Jiale Chen, Hui Gong, Xiangning Li |
| المصدر: | International Journal of Molecular Sciences, Vol 25, Iss 12, p 6504 (2024) |
| بيانات النشر: | MDPI AG |
| سنة النشر: | 2024 |
| المجموعة: | Directory of Open Access Journals: DOAJ Articles |
| مصطلحات موضوعية: | paraformaldehyde fixation, cross-link reversal, RNA sequencing, full-length transcripts, protein-coding genes, non-coding RNAs, Biology (General), QH301-705.5, Chemistry, QD1-999 |
| الوصف: | Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the amine groups of biomolecules to form chemical cross-linking, which preserves RNA within the tissue. This has great prospects for RNA sequencing to characterize the molecular underpinnings after anatomical and pathological observations. However, RNA is inaccessible due to cross-linked adducts forming between RNA and other biomolecules in prolonged PFA-fixed tissue. It is also difficult to perform reverse transcription and PCR, resulting in low sequencing sensitivity and reduced reproducibility. Here, we developed a method to perform RNA sequencing in PFA-fixed tissue, which is easy to use, cost-effective, and allows efficient sample multiplexing. We employ cross-link reversal to recover RNA and library construction using random primers without artificial fragmentation. The yield and quality of recovered RNA significantly increased through our method, and sequencing quality metrics and detected genes did not show any major differences compared with matched fresh samples. Moreover, we applied our method for gene expression analysis in different regions of the mouse brain and identified unique gene expression profiles with varied functional implications. We also find significant dysregulation of genes involved in Alzheimer’s disease (AD) pathogenesis within the medial septum (MS)/vertical diagonal band of Broca (VDB) of the 5×FAD mouse brain. Our method can thus increase the performance of high-throughput RNA sequencing with PFA-fixed samples and allows longitudinal studies of small tissue regions isolated by their in situ context. |
| نوع الوثيقة: | article in journal/newspaper |
| اللغة: | English |
| تدمد: | 1422-0067 1661-6596 |
| Relation: | https://www.mdpi.com/1422-0067/25/12/6504; https://doaj.org/toc/1661-6596; https://doaj.org/toc/1422-0067; https://doaj.org/article/9957ffb4d1a1476dbfb6c3fe1f59e5a0 |
| DOI: | 10.3390/ijms25126504 |
| الاتاحة: | https://doi.org/10.3390/ijms25126504 https://doaj.org/article/9957ffb4d1a1476dbfb6c3fe1f59e5a0 |
| رقم الانضمام: | edsbas.E1EEA48E |
| قاعدة البيانات: | BASE |
Full Text Finder | تدمد: | 14220067 16616596 |
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| DOI: | 10.3390/ijms25126504 |