Stability And Integrity Of RNA In Yak Milk Under Different Processes And Treatments

More and more studies have found that there is a small amount of RNA in dairy products,though RNA is unstable due to its single-stranded structure.These RNA is resistant to adverse environments and are more stable.Therefore,the yak milk samples of different months have been processed in different ways in this study to research the stability and integrity of RNA in yak milk.And through high-throughput sequencing,microRNAs in yak exosomes at the early lactation and at the mid lactation have been sequenced and compared.(1)The yak milk was treated differently,40,50,60,70,80,90 heating for 30 ℃min and-20℃ conserving for 360,330,300,270,240,210 d,the concentration and purity of total RNA and exosomal RNA were significantly different from which in fresh yak milk(P<0.05).The amplified bands of the SCD1 gene of total RNA in yak milk samples being heated at 80,90℃ were not clear and neat.Under the other conditions,the total RNA and exosomal RNA of yak milk showed clear amplified bands,which could meet the need of deep gene research.(2)The yak milk was processed differently,yak milk freeze-dried powder,yak cheese,yak yogurt and yak milk powder,the concentration and purity of total RNA and exosomal RNA were significantly different from which in fresh yak milk(P<0.05).The total RNA of yak milk freeze-dried powder and exosomal RNA of the four kinds of dairy products appeared clear and neat bands when the SCD1 gene was amplified,which could meet the need of further gene research.(3)Using BO/YA-FW,BO/YA-RV and BO-FW primers to amplify the ND1 gene in milk exosomes,the composition of bovine milk in yak milk,pasteurized yak milk and yak milk powder could be detected,and the detection limit was 0.1%.The mRNA could be stable because of the protection of exosomes through deep processing or treatments,which provided a new idea for adulteration detection of yak milk.(4)Two miRNA libraries were constructed using RNA in exosomes isolated from Fst-exs and Mid_exs using high-throughput sequencing,12,202,741 and 11,214,499 clean reads were obtained respectively which accounted for 98.16% and 99.07% of their raw reads respectively.3 miRNAs,1 mi RNA* chain and 3 pre-miRNAs were predicted in total.251 differential expressed miRNAs were screened out from 2 samples.There were 78 significantly up-regulated miRNAs and 89 significantly down-regulated miRNAs at Mid_esx compared with Fst_exs.The top 20 of the most significant differences in expression were related to the adipocyte differentiation and immune regulation.Finally the GO enrichment showed that the target genes of differentially expressed miRNAs mainly involved in a variety of biological processes such as cell migration and regulation of cell migration.KEGG enrichment showed that the ribosome pathway(Ribosome)was a significant enrichment term,enriching to 94 candidate target genes.