Streamlined Full-Length Total RNA Sequencing of Paraformaldehyde-Fixed Brain Tissues

Author:

Ji Bingqing12,Chen Jiale12,Gong Hui123,Li Xiangning34

Affiliation:

1. Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China

2. MoE Key Laboratory for Biomedical Photonics, Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China

3. Research Unit of Multimodal Cross Scale Neural Signal Detection and Imaging, HUST-Suzhou Institute for Brainsmatics, JITRI, Chinese Academy of Medical Sciences, Suzhou 215125, China

4. Key Laboratory of Biomedical Engineering of Hainan Province, School of Biomedical Engineering, Hainan University, Haikou 570228, China

Abstract

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.

Funder

Major Program of National Natural Science Foundation of China

National Natural Science Foundation of China

Publisher

MDPI AG

Reference68 articles.

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