The Extraction And Separation Of MiRNA In Animal Tissue And Its Structure And Function Prediction

MicroRNA (miRNA) are small non-coding RNA with a length about18~25nt,existing in eukaryotic cells and regulating gene expression by complete or incompletebinding to3’ untranslated regions of target mRNA, thereby leading to inhibition oftranslation or degradation of mRNA. Since the first miRNA was found in1993, theresearch on miRNA has been a research hot of life science. In recent years, with thedeepening of the research, people began to realize that miRNA has a broad role in theregulation of gene expression. Currently, the research fields of miRNA includeexpression profiling and functional analysis of miRNA. The miRNA has also becomeresearch focus of clinical medicine in recent years, miRNA are related to manydiseases due to its regulatory effect, so the miRNA research on detection andtreatment of disease will make breakthrough in the future.Based on the structure character of poly(A) in mRNA, This research lookedforward to screen unkown miRNA through under steps. Firstly, poly(A) was linked tomiRNA3’, the linker was then linked to poly(A)–miRNA5’; secondly,poly(A)–miRNA–linker was converted to cDNA by reverse transcription with oligo(T)as primers and then amplified by PCR; thirdly, target fragment with appropriate lengthwas selected through electrophoresis and transformed to Escherichia coli, signal clonebacterium solution after transformation was identified by stagger PCR, the targetfragment was futher analysed by DNA sequencing. According to this method, we havescreened known miRNA, so it is effective and applicative for screen of miRNA.In this study, not retrieved in miRNAbase and Rfam, one suspected miRNA with18~25bases has been selected from the mice liver and domestic fowl liver,respectively. BLAST comparison analysis in NCBI indicated that the sequence ofmiRNA screened from mice liver was located at gene which could transcribe tomRNA in mice embryonic stem cell, it probably involved regulation and control ofembryonic stem cell differentiation; the sequence of miRNA screened from domesticfowl liver was located at E-selectin gene3′, it probably involved regulation andcontrol of E-selectin expression. The secondary structure of pre-miRNA predicted viaRNAdraw indicated that they all could form stem–loop region where miRNAsequence was located, according with the structure character of miRNA. Thus, these two miRNAs was probably unkown miRNA, but their function analysis will beattributed to further study.