The Research On Acquisition And Preparation Of Functional MicroRNA In Food

Background and purpose:Accounting for about98%of the non-coding RNA genes in the entire genome of the plants and animals, in particular microRNA burden of the regulation of gene expression and function. Plasma, there are several mature miRNA and miRNA extraction and separation than some high molecular weight RNA stability. It has been believed that the ingestion of nucleic acid components in the digestive tract decomposition of single nucleotide even broken down into the base after intestinal absorption. From this perspective, the study of feeding miRNA on the role of the body and health impacts, the first is to get a lot of microRNA raw feeding. If able to collect natural and stable microRNAs on the the study feeding miRNA functional properties of great theoretical and practical significance.Methods:Surimi production wastes as raw materials extraction and alcohol precipitation concentrated, polyacrylamide gel electrophoresis in line with the amount of RNA microRNA molecular size. Then the gene DNA sequence of a known sequence of mature M168a (microRNA) to form a combination with the T7promoter T7promoter in the role of the T7RNA polymerase, transcription of RNA as internal standard electrophoresis enrichment molecule. Gel film can be used to isolate design a special enrichment electrophoresis device, between the positive and negative. And then with FITC fluorescence-labeled synthetic DNA molecules as internal standard, and explore the liquid electrophoresis enrichment conditions. Under different electrophoretic fluid conditions, different electrophoresis time is detected, which can be enriched in the amount of the fluorescent molecules, and then to poly acryl amide gel electrophoresis, inspection enriched fluorescence labeled comprehensive. Rely worked out of the liquid electrophoresis conditions, small RNA (M168a) as an internal standard to verify the feasibility of the electrophoresis conditions, with the Nano Drop Ultramicro UV spectrophotometer M168a is enriched to a concentration. Surimi production waste, electrophoresis to collect a result of the different times, while the surimi production scrap M168a internal standard substance. The Nano Drop Ultramicro UV spectrophotometer detection increases with time content. The effective electrophoretic membrane area large enough to meet our collection of the target moleculeResults:With the low toxicity of the extract (containing a variety of anionic surface active agent), was extracted in a water bath of100℃15-20min, obtained compared to ordinary methods of RNA isolation macromolecular RNA content is low, the small RNA molecules extracted total extracts ofthe high ratio of the amount column, can basically meet the requirements of extracting microRNA experiments. In food processing waste system small and medium RNA than macromolecular RNA more stable. The need for effective methods to increase the concentration of the unit volume microRNA weak content of about25nt microRNA enrichment is to be resolved.The pilot plant, liquid gel film electrophoresis gel membrane between the cathode smaller than can be replaced. Groping experiment conditions with different kinds of nucleic acid molecule as an internal standard, determined at a higher voltage300V, TBE electrophoresis buffer with a lower concentration, in0.2xTBE or lower concentrations, was subjected to electrophoresis. Positive electrode volume is reduced to a10-fold concentrated as the negative electrode1/10,40min of electrophoresis, the small RNA through the membrane equivalent. Surimi rinsing effluent complex containing a nucleic acid component, in addition to the small RNA molecules to migrate to the other end of the film, the degradation of macromolecules was also migrated through the membrane.Use of constitutive T7promoter, the T7RNA polymerase,1.6μM of the DNA as a template, was synthesized with a short promoter sequence microRNAs. Cyclized by the chain of the active center with a DNAzyme (8-17type), the control of the nuclease activity of cutting the RNA phosphodiester bond, provided the basis for the mature microRNA slowly.Conclusions:The preliminary study on the acquisition and preparation of microRNA low toxicity of the extract to extract enriched microRNAs synthetic small RNAs using T7RNA polymerase transcription system, specific conclusions are as follows:The low toxicity of the extract, the extract small molecule RNA share of the total extract higher compared to conventional extraction methods can basically meet the requirements of extracting microRNA experimental. Relatively stable in the liquid waste system small and medium RNA.This experiment successful design of a small pilot plant liquid gel film electrophoresis, the gel film between the positive and negative replacement. Determined at a higher voltage300V, the lower the concentration of the electrophoresis buffer TBE, subjected to electrophoresis. Small volume experiments, using the internal standard molecules test,40min enrichment concentration increased gradually, the surimi rinsing waste liquid, in addition to the small RNA molecules to migrate to the other end of the film, the degradation of macromolecular nucleic acid also migrated through the membrane. At the same time, if you can be prepared enough known size and sequence of small RNA as an internal standard electrophoresis molecules, can also be measured electrophoresis enrichment efficiency of small RNA.This paper composed of type T7promoter, to a DNA template of about1.6μμM, in the T7RNA polymerase under the action, were synthesized with a short promoter sequence microRNAs. Cyclized DNAzyme (8-17type) the activity of the phosphodiester bond cleave RNA is restricted, for mature microRNA slowly in the body provide a basis.