Microfluidic Microarray Screening Mouse Tissue-specific Expression Of MiRNAs

Non-coding RNA is increasingly becoming a hot international study, which not only can regulate gene expression, but also closely related to cancer. As a large class of non-coding RNA, tissue-specific expression is one important characteristic of miRNA expression. Identifying tissue-specific miRNAs is the first step toward understanding the biological functions of miRNAs. In addition, in different developmental phase and between male and female mouse, miRNA has a different expression profile. In this paper, we carry out the work mainly from the following three aspects:(1) To collect miRNAs of 18 kinds of tissues of mouse: for the preparation of screening of tissue-specific expression of miRNAs, we obtain a large number of papers (about 300) relevant to the study of mouse miRNA by retrieving PubMed, and collect miRNAs that expressed in various tissues of mouse at different developmental stages as well as different gender and a variety of other conditions, through intensive reading literature.(2) Use microfluidic microarray technology to screenin miRNAs of mouse six kinds of tissues: microfluidic chip technology has characteristics of good flexibility, high degree of automation, wide application and low sample consumption, no cross-contamination and good result reproducibility, the application of the technology provides a convenient, cost-effective method for us to screen gene expression by high-throughput. We dissect four C57BL/6 mice to obtain six kinds of tissues: cerebrum, cerebellum, liver, lung, testis and kidney. And then, total RNA are extracted after mixing equal tissue from four mice respectively. Recover high-quality of small RNA (smRNA, 10-50 nt) by cutting urea-PAGE gel. After that, 5'and 3'RNA adapters are ligated to the both ends of smRNA respectively, and RT-PCR is used to build smRNA library at last (smRNA library contains miRNA). MiRNA library is recovered by cutting native PAGE gel according to the band, and finally, signal primer PCR is used to label miRNA library with fluorescent. Then we use microfluidic chip to detect miRNA expression in different tissues. It has a good effect to detect low expression of miRNAs with this method. To improve detection accuracy, we take only those miRNAs detected in at least four out of five averages arrays for the credible result. We find that the cerebrum contains 246 miRNAs, cerebellum contains 291 miRNAs, liver contains 209 miRNAs, lung contains 230 miRNAs, testis contains 84 miRNAs, kidney contains 40 miRNAs.(3) Combination of (1) and (2) to analyze mouse six different tissues of tissue-specific expression miRNAs: by comparing six tissues miRNAs detected with microfluidic chip and screening, except testis, we get tissue-specific expression of miRNAs of other five tissues. In order to further obtain more reliable result, we make a further comparison with the expression of miRNAs under different conditions in mouse tissues based on literature. As a result, we find that some miRNAs are still expressed in other tissues. Indicating that, informatics combination of experiment is a powerful research tools.Microfluidic chip technology combination of bioinformatics, not only can avoid a large number of unnecessary experiments, saving cost, but also can speed up the pace of scientific research and enhance scientific research level. Powerful combination of both will also bring new opportunities for the development of biology.