Development Of Fragmentation Method For Noncoding RNA And Its Verification

Non-coding RNAs (ncRNAs) are functional RNAs without conservative open reading frames that do not code proteins. It is necessary to develop an effencient screening method for ncRNA research. The long ncRNAs could form a complex secondary structure under natural conditions, which is the main factor that affects microarray hybridization efficiency. In order to reduce the secondary structure effect, RNA is often haven to be fragmented prior to hybridization. The fragmentation of RNA (20-200 nt) can avoid the negative impact of secondary structure and increase the hybridization efficiency.The total RNA of cell line was used to optimize the RNA fragmentation mothed by using three metal ions. The results showed that Mg²⁺ could fragment the total RNA at the size of 20-200 nt; The capacity of fragmenting RNA by Zn²⁺was closely related to the temperature, at 70°C, generating 20-200 nt RNA fragments; at 95°C generating 20-60 nt RNA fragments. Na+ could fragment RNA at a relatively lower temperature, at 37°C could produce RNA fragments at sizes of 20-200 nt, and at 47°C could produce RNA fragments at sizes of 20-60 nt. This method can provides enough fragmented RNA quickly to hybridize with one genome-scale microarray.A method for fluorescence labling of fragmented RNA was also developed. RNA fragmentation regeant can fragment the RNA randomly. The fragmented RNA may have phosphate group at either 5'end or 3'end. Under natural conditions, 3'end of RNA is the hydroxyl group and 5'end is the phosphate group, the fragmentation RNA should be treated by the process of dephosphorylation at frist. And then the fragmented RNA shoud be treated by the process of phosphorylation. The processed RNA can be ligated with double strands RNA adaptor at the same time. In order to improve the ligation efficiency, the size of fragmented RNA should be in a narrow extent. The homogeneous fragmented RNAs could be obtained after recovered from PAGE gel. After ligating the RNA adaptor, the fragmented RNA could be carried out to PCR amplication and labeled by Cy⁵ fluorescent dyes.The tiling array of Riboswitch9 study shown that the processing technique of fragmentation could enhance the RNA microarray signal. It was necessary to fragment ncRNA to reduce the secondary structure effect. In this experiment, the method of processing ncRNA sample was established and provided certain technical support for ncRNA research.