5-hydroxymethylcytosine is detected in RNA from mouse brain tissues.
5-hydroxymethylcytosine (5hmC) is considered as a novel DNA modification and plays an important role in cancer, stem cells, and developmental diseases. In this study, we demonstrated the existence of RNA 5hmC modification in mouse brain RNA by using a dot blot analysis method. Our data indicated that 5hmC modification in RNA samples was less than that in DNA samples. Further, we optimized the conditions for 5hmC detection in RNA samples such as DNase treatment, denature reagents, denature time, sample air-dry time, and the cross-linking time between RNA and membrane. Our results demonstrated that DNase treatment and denature reagents were two important factors that affected the 5hmC detection in RNA samples. By using the optimal conditions for RNA 5hmC detection, we found that the brainstem, the hippocampus, and the cerebellum had high levels of 5hmC modification and 5mC modification in RNA. Finally, we found that RNA 5hmC modification decreased in MPTP-induced Parkinson's disease model in mice. These suggest that 5hmC modification in RNA might play an important regulative role on protein or microRNA expression in these brain tissues. Because DNA 5hmC modification plays an important role in neural differentiation and development as well as neurological diseases, the significance of 5hmC modification in RNA in different neurological diseases needs further investigation. In summary, our study demonstrated for the first time the abundance of 5hmC modification in brain RNA by using a dot blot analysis method and proved that dot blot analysis is a useful method for 5hmC detection in RNA samples.
Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou City, Jiangsu, China; Institute of Neuroscience, Soochow University, Suzhou City, Jiangsu, China.
Zhigang
Z
Miao
Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou City, Jiangsu, China.
Ning
N
Xin
Institute for Fetology, the First Affiliated Hospital of Soochow University, Suzhou City, Jiangsu, China.
Bin
B
Wei
Department of Emergency, Emory University Hospital, Atlanta, GA, USA; Department of Biochemistry, Franklin College of Arts and Sciences, University of Georgia, Athens, GA, USA.
Xiaodong
X
Hua
Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou City, Jiangsu, China; Institute of Neuroscience, Soochow University, Suzhou City, Jiangsu, China.
Gaocai
G
Zhang
Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou City, Jiangsu, China; Institute of Neuroscience, Soochow University, Suzhou City, Jiangsu, China.
Cuihua
C
Leng
Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou City, Jiangsu, China; Institute of Neuroscience, Soochow University, Suzhou City, Jiangsu, China.
Chenyu
C
Zhao
Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou City, Jiangsu, China; Institute of Neuroscience, Soochow University, Suzhou City, Jiangsu, China.
Di
D
Wu
Department of Neurology, Suzhou Kowloon Hospital, Suzhou City, China.
Jizhen
J
Li
Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou City, Jiangsu, China; Department of Neurology, the Affiliated Hospital of Xuzhou Medical College, Xuzhou City, Jiangsu, China.
Wei
W
Ge
Institute for Fetology, the First Affiliated Hospital of Soochow University, Suzhou City, Jiangsu, China. Electronic address: sunmiaosun@yahoo.com.
Miao
M
Sun
Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou City, Jiangsu, China; Institute of Neuroscience, Soochow University, Suzhou City, Jiangsu, China. Electronic address: xingshunxu@suda.edu.cn.
Xingshun
X
Xu
D044503:5-Methylcytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D006358:Hot Temperature; D015151:Immunoblotting / Q000379:methods; D020267:MPTP Poisoning / Q000378:metabolism; D008297:Male; D008813:Mice, Inbred ICR; D009043:Motor Activity; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D012313:RNA / Q000378:metabolism*; D011897:Random Allocation; D013997:Time Factors
C011865:5-hydroxymethylcytosine; D012313:RNA; D044503:5-Methylcytosine; D004247:DNA
5-Hydroxymethylcytosine; 5-Methylcytosine; Dot blot; Method; RNA