Cloning MicroRNA And Its Target Gene Prediction In Chicken’s Liver

MicroRNAs(miRNAs)are non-encoded small RNAs which discovered in recentyears,about18-24nucleotides in length.They mediate the post-transcriptional genesmodulation generally and regulate various physiological processes in the body.miRNAs areclosely related to the occurrence and development of human diseases such as tumour.Studiesfound that there are miRNAs in the blood circulation,and circulated miRNAs can act as anew non-invasive turnout diagnosis biomarkers.Currently the discovery of unknown miRNAsequences mainly base on bioinformatics prediction, direct cloning method was rarely.Switching mechanism at5’end of the RNA transcript(SMART) method has been used toobtain new microRNAs sequences from chicken’s liver. In the experiment, RNA fractionswere extracted and polyadenylated by poly(A) polymerase at first; then, cDNA library whichwas suitable for PCR has been constructed by SMART reverse transcript technology; afterthat, the target cDNA fragments were recovered and cloned into vector for sequencing, andthree microRNAs were discovered. Sequence analysis showed that all of them were notexisted in miRbase,but their precursors can form stem-loop structures,therefore thesequences can be classified into new microRNAs.Next,three commonly used target gene prediction software which are miRanda、TargetScan and MirTarget have been used to predict the target gene of the three newmicroRNAs.And then,using target gene sequences which three software can alsopredicted to analysis biology function of three microRNAs.The result showed that targetgene significantly enrichment in102GO classification and13signal path.Most targetgenes involved in many life activities which include signal transduction, metabolism, celladhesion and across the plasma membrane,and also closely related to the growth ordifferentiation of embryonic stem cell. The result provide methodology reference for thedirect cloning of microRNA and microRNA target gene function analysis.