The Methods Of MicroRNAs Isolation And Purification

MicroRNAs are the conserved non-coding small RNAs family of18-24nucletides (18-24nt) in length. In eukaryotes, they regulate the expression of genes by complementary association with target mRNA at the translational level. The researches about microRNAs suggest that microRNAs are regulating functional on cell multiplication, differentiation apoptosis and the developing of tumor. Although there are many researches on microRNAs and their functions, the technoledge of microRNAs isolation and purification was ineffective, especially in serum microRNAs, which needs to be improved.In the study of microRNA isolation and purification, we choose mice liver tissue and serum as the experimental materials.First, we extracted and isolated microRNA from mice liver tissue. We extracted total RNA from mice liver by RNAiso Plus, and then used poly (A) method:Add a poly (A)-tail to3’end of microRNA by poly (A) polymerase and a L2linker to5’end. After RT-PCR by Oligo(dT), the cDNA library was cloned into T vector, then we chose the positive clones to sequence.The sequencing results showed that4microRNAs could be identified from the mice liver cDNA library, analysis results were as follows:1. Sequence No.7and No.8were sense strand and antisense strand of miR-122;2. Sequence No.3and No.6did not match the miRBase, which might be the unknown small RNAs. We predicted the possible structure of these2RNA, found they both could form stem-loop structures, which was a structure character of microRNA. Sequence No.3and No.6were submitted to GeneBank:BankIt1498739Seql JQ343827and BankIt1498739Seq2JQ343828. BLAST analysis of the two showed Sequence No.3was mapped to apolipoprotein B (Apob) gene and might regulate Apob gene; Sequence No.6was mapped to28S ribosomal RNA and might associate with regulating ribosomal RNA gene.Based on the mice liver experiment, we studied isolation and purification of human serum microRNAs. We extracted total RNA from human serum by RNAiso Plus, and selected3methods to isolate microRNAs: 1. Poly (A) method:Based on the isolation and purification of mice liver, we obtained cDNA library, cloned and sequenced target sequences;2. Linker method:2synthetic oligonucleotides of known sequence linkers, L1and L2, were separately ligated on the3’and5’ends by T4RNA polymerase. Prime matched linker L1was used to reverse RNA into cDNA library, then cloned and sequenced them;3. Dra I method:3’end poly (A)-tail was polyadenylated by poly (A) polymerase, and reversed RNA into cDNA library. Linker P16was ligated on the3’end by T4RNA polymerase. After PCR, we used Dra I restriction enzyme to cut off incorrect linking, linker was ligated with poly (A)-tail. PCR cloned and sequenced.The results were as follows:1. Poly (A) method:PAGE showed that on110bp there were objective sequences, the clones were false-positive results that linker L2ligeted with poly (A)-tail;2. Linker method:PAGE showed that on110bp there were not objective sequences, on90bp there was a band. The sequencing results showed that linker L2ligeted with poly (A)-tail, so there was no small RNA ligated with linkers;3. Dra I method:PAGE showed that on90bp there were objective sequences, the clones were false-positive results that linker L2ligeted with poly (A)-tail.In summary, we obtained4microRNAs in mice liver,2of them unknown sequences were submitted into GeneBank. Based on it, we primarily explored the methods of human serum extraction and isolation. All of these3methods, poly (A) method, linker method and Dra I method, have certain shortcomings and need to be improved. The study gave the basis to methods of extraction and isolation in human serum.