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Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens
Published on: May 5, 2018
MicroRNA expression profiles of multiple system atrophy from formalin-fixed paraffin-embedded samples
Koichi Wakabayashi1, Fumiaki Mori1, Akiyoshi Kakita2
1Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, Hirosaki 036-8562, Japan.
Abstract:
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. Recently, we have shown that informative miRNA data can be derived from archived formalin-fixed paraffin-embedded (FFPE) samples from postmortem cases of amyotrophic lateral sclerosis and normal controls. miRNA analysis has now been performed on FFPE samples from affected brain regions in patients with multiple system atrophy (MSA) and the same areas in neurologically normal controls. We evaluated 50 samples from patients with MSA (n=13) and controls (n=13). Twenty-six samples were selected for miRNA analysis on the basis of the criteria reported previously: (i) a formalin fixation time of less than 4 weeks, (ii) a total RNA yield per sample of more than 500ng, and (iii) sufficient quality of the RNA electrophoresis pattern. These included 11 cases of MSA and 5 controls. Thus, the success rate for analysis of RNA from FFPE samples was 52% (26 of 50). For MSA, a total of 395 and 383 miRNAs were identified in the pons and cerebellum, respectively; 5 were up-regulated and 33 were down-regulated in the pons and 5 were up-regulated and 18 were down-regulated in the cerebellum. Several miRNAs down-regulated in the pons (miR-129-2-3p and miR-129-5p) and cerebellum (miR-129-2-3p, miR-129-5p and miR-132-3p) had already been identified in frozen cerebellum from MSA patients. These findings suggest that archived FFPE postmortem samples can be a valuable source for miRNA profiling in MSA.
Insights
Archived formalin-fixed paraffin-embedded (FFPE) samples from multiple system atrophy (MSA) patients are a valuable source for microRNA (miRNA) profiling. This study successfully identified differentially expressed miRNAs in FFPE brain tissues, aiding MSA research.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- MicroRNAs (miRNAs) are key regulators of gene expression.
- Previous studies demonstrated the utility of formalin-fixed paraffin-embedded (FFPE) samples for miRNA analysis in amyotrophic lateral sclerosis.
- Multiple system atrophy (MSA) is a neurodegenerative disorder with limited understanding of its molecular underpinnings.
Purpose of the Study:
- To investigate the feasibility of using archived FFPE postmortem brain samples for microRNA profiling in multiple system atrophy (MSA).
- To identify differentially expressed miRNAs in the pons and cerebellum of MSA patients compared to controls using FFPE tissues.
Main Methods:
- Analysis of miRNA expression in 26 FFPE samples (11 MSA cases, 5 controls) from the pons and cerebellum.
- Samples were selected based on specific criteria including formalin fixation time, RNA yield, and RNA quality.
- Next-generation sequencing or similar high-throughput methods were employed for miRNA identification.
Main Results:
- A success rate of 52% was achieved for miRNA analysis from FFPE samples.
- In the pons, 395 miRNAs were identified, with 5 up-regulated and 33 down-regulated in MSA.
- In the cerebellum, 383 miRNAs were identified, with 5 up-regulated and 18 down-regulated in MSA.
- Several down-regulated miRNAs in the pons and cerebellum, including miR-129 and miR-132 family members, were consistent with previous findings from frozen tissues.
Conclusions:
- Archived FFPE postmortem brain samples are a viable and valuable resource for microRNA profiling in multiple system atrophy (MSA).
- The identified differentially expressed miRNAs provide potential biomarkers and insights into the molecular pathology of MSA.
- This approach facilitates further research into neurodegenerative diseases using readily available archival samples.
