Primary Research On Enrichment Technology For Typical MiRNA (miR-206) In Paralichthys Olivaceus

MicroRNAs (miRNAs) are a type of endogenous RNAs (~22nt) that playimportant roles on gene expression and regulation of animals and plants cells bydestroying or suppressing targeting RNAs. Thus, researches related to miRNA haveattracted more and more attentions. Resently, a research showed that the ingestedmiRNA (miR-168a) from rice could regulate the physiologicalaction of mice. Theresult indicated that miRNAs were brand new bio. However, the mechanism of thiscross “kingdom” regulation was still unclear. More and more miRNAs materials wereconsumed in the related researches. At present, chemosynthesis is the availablemethod to obtain miRNAs. The method is only suitable for preparation of “microgram”miRNAs, and usually the price is super high (more than thousands of yuan per mol).Moreover, toxic chemical reagents are often employed during synthesis. Therefore,the lack of preparation method for miRNA impedes the further researches on miRNA,especially study on the regulation function of miRNA from food which need a lot ofnon-toxic miRNAs material. Therefore, it is very important to find an efficient,convenient and cheap way to produce the miRNAs. This study aimed to enrich theedible miRNAs (miR-206) in Paralichthys olivaceus. RT-PCR method wasestablished to determine miRNAs. Gradient concentration ethanol precipitation andsepharose gel electrophoresis were investigated to enrich miRNAs from fish wastewater.A reverse transcription quantitative PCR detection method was established firstly.Two kinds of stem loop primers for miR-168a and miR-206were designed. Thedetection concentration range and sensitivity were evaluated for both miR-168a andmiR-206. Results showed that this method had a high specificity, after38-cycle-PCRamplification nonspecific amplification was not detected. The linear range was wide,the standard curve that established with miRNA quantitative PCR reactionconcentration and Ct values can detect the concentration of sample from10nM to1 fM, and with the R2higher than0.99. The result of miR-206determination showedthat both Paralichthys olivaceus skeletal muscle and the supernate of minced fishwater contain considerable raw miRNAs. The fish is an ideal material to extract orenrich miR-206. The minced fish water was processed used different way, such asultrasonic concussion and freeze thawing. The concentration of miR-206insupernatant after the ultrasonic processing was twice more than the miR-206in freezethawing processing group. Comparing the two pre-treatment, ultrasonic methodshowed to be an ideal way to break cells and release miRNAs.To enrich miRNAs in fish, miR-206solution with fixed concentration andminced fish solution were treated with different concentrations of ethanol. Resultsshow that in minced fish water, the content of miRNA in precipitation wassignificantly increased with the increase of ethanol concentration. When treated with70%ethanolmore than95%of miRNAs in the minced fish solution were precipitated.According to the results of gradient ethanol precipitation, minced fish solution wastreated with concentration of10%/30%/50%ethanol step by step, finally more than60%of miR-206was enriched in the precipitation. It could be a good material forfurther purification.For film electrophoresis enrichment of nucleic acid, a82bp DNA fragment wasfirst used to optimize the membrane electrophoresis conditions, including voltage(electric field), electrophoresis buffer concentration (ionic strength), gel membraneconcentration, etc. It was showed that under the same buffer concentration (1xTBE) and film concentration (2%), the higher voltage, the nucleic acid accumulatedfaster with the increase of votage. rate. When used150V voltage, the nucleic acidaccumulation rate was eight times faster than that of50V voltage. Buffer heating ratewas in direct proportion to the voltage, when used150V voltage the heating rate was1oC/min, and100V was0.5oC/min,50V was0.1oC/min. When using the samevoltage (100V) and film concentration (2%) with0.5x,1x,1.5x TBE aselectrophoresis buffer, the ionic strength was associated with electrophoresisenrichment efficiency, when used0.5x TBE as electrophoresis buffer, the positivenucleic acid concentration was only sixty percent of1.5x TBE after45min. The buffer was obviously wormed up when improved the ion concentration,1.5x TBEheating rate was0.4oC/min,1.0x and0.5x TBE heating rate were0.2oC/min and0.1oC/min respectively. Gel concentration tests showed that nucleic acid swim fastestwhen using1.5%gel. The speed was nearly three times as higher as2.5%gel.Concentration of1.5%gel enriched most of the DNA after10min, save nearly10min compared with the other two gel concentrations. When using the optimizedconditions to enrich282bp of DNA, the DNA concentration was six times higherthan the initial sample. The minced fish solution was treated with ethanolprecipitation and membrane electrophoresis, finally got a large amount of of miRNAswith a relatively high quality. Minced fish water or the precipitate after ethanolprecipitation treatment, continue to use the film electrophoresis for enrichment ofmiRNAs, namely more than9fmol miR-206were enriched within0.5h. Comparedwith the minced fish water without ethanol precipitation and protease treatment andenriched with film electrophoresis directly, the miR-206content in former sample wasbeyond the latter nearly30times.