本文選題：變應(yīng)性鼻炎　切入點(diǎn)：RNA-seq測序　出處：《中南大學(xué)》2014年碩士論文

【摘要】：目的 探討長鏈非編碼RNA (Long Noncoding RNA, lncRNA)在變應(yīng)性鼻炎與正常鼻粘膜中是否存在表達(dá)差異,并篩選出差異顯著的長鏈非編碼RNA。 方法 1)分別提取變應(yīng)性鼻炎和正常鼻粘膜組織總RNA,質(zhì)量檢測后合成雙鏈cDNA, PCR擴(kuò)增建立cDNA文庫,用Illumina HiSeqTM2000測序得到原始序列數(shù)據(jù)。運(yùn)用軟件Tophat、Cufflinks構(gòu)建轉(zhuǎn)錄本。運(yùn)用blast與non-coding RNA、kegg、nr、cog、swissprot等數(shù)據(jù)庫比對,識別出已知的lncRNA和：mRNA;利用CPC基于framefinder和blast進(jìn)行SVM預(yù)測,得到預(yù)測的新的非編碼RNA。 2)運(yùn)用RPKM法分別計算lncRNA和mRNA表達(dá)量,基于差異倍數(shù)運(yùn)用聚類分析篩選出多對樣本均有差異表達(dá)的lncRNA和mRNA。 3) qRT-PCR對部分差異表達(dá)的長鏈非編碼RNA進(jìn)行驗證。 4)運(yùn)用關(guān)系式數(shù)據(jù)分析系統(tǒng)RDAS計算差異表達(dá)的lncRNA和所有mRNA的關(guān)聯(lián)性,得到與差異表達(dá)的lncRNA共表達(dá)的mRNA。 5)運(yùn)用數(shù)據(jù)庫KEGG、BioCarta對共表達(dá)的mRNA進(jìn)行Pathway分析,得到共表達(dá)的mRNA參與的生物學(xué)途徑。結(jié)合生物學(xué)途徑結(jié)果和關(guān)聯(lián)性結(jié)果了解lncRNA的調(diào)控作用。 結(jié)果 1)本研究首次應(yīng)用RNA-Seq測序技術(shù)得到變應(yīng)性鼻炎和正常鼻粘膜的lncRNA表達(dá)譜和mRNA表達(dá)譜。 2)3對樣本中均有差異表達(dá)的lncRNA共173種,120種上調(diào),53種下調(diào)；已知的lncRNA63種,49種上調(diào),14種下調(diào)；新的lncRNA110個,71種上調(diào),39種下調(diào)。3對樣本中均有差異表達(dá)的mRNA共437種,其中365種高表達(dá),72種低表達(dá)。 3)經(jīng)qRT-PCR驗證長鏈非編碼RNA NONHSAT250987, NONHSAT176469、NONHSAT309646、NONHSAT140585、 NONHSAT201926表達(dá)呈下調(diào)趨勢；長鏈非編碼RNA NONHSAT26156、NONHSAT292828、NONHSAT312751、 NONHSAT157109表達(dá)呈上調(diào)趨勢；結(jié)果與RNA-seq預(yù)測結(jié)果相一致。 4)關(guān)聯(lián)性分析得到與85種差異表達(dá)的lncRNA共表達(dá)的mRNA,共379種。 5)經(jīng)Pathway分析得到共表達(dá)的mRNA的17條生物學(xué)途徑。 結(jié)論 變應(yīng)性鼻炎患者鼻粘膜lncRNA的表達(dá)與正常鼻粘膜比較存在顯著性差異。
[Abstract]:PurposeTo investigate the difference of long chain noncoding RNA long Noncoding (LNC RNAs) between allergic rhinitis and normal nasal mucosa, and to screen out the significant difference of long chain non coding RNA (LNAs) between allergic rhinitis and normal nasal mucosa.Method1) Total RNAs were extracted from allergic rhinitis and normal nasal mucosa respectively. After quality detection, double-stranded cDNAs were synthesized, cDNA library was established by PCR amplification, and original sequence data were obtained by Illumina HiSeqTM2000 sequencing.The transcripts were constructed by using the software Tophatine Cufflinks.By comparing blast with non-coding, we can identify the known lncRNA and 1: mRNAs, and use CPC to predict SVM based on framefinder and blast, and get a new noncoding RNAs.2) the expression of lncRNA and mRNA were calculated by RPKM method, and the lncRNA and mRNA with different expression were selected by clustering analysis based on the difference multiple.3) qRT-PCR was used to verify the partial differential expression of long chain non-coded RNA.4) using the relational data analysis system (RDAS) to calculate the correlation between the differentially expressed lncRNA and all mRNA, and to obtain the mRNAs coexpressed with the differentially expressed lncRNA.5) the co-expressed mRNA was analyzed by Pathway using the database KEGGG BioCarta, and the biological pathway of co-expressed mRNA was obtained.Combined with biological pathway results and correlation results to understand the regulatory role of lncRNA.Result1) the lncRNA and mRNA expression profiles of allergic rhinitis and normal nasal mucosa were obtained by RNA-Seq sequencing for the first time.(2) there were 173 species of lncRNA with different expression and 53 down-regulated species of lncRNA, 49 species of known lncRNA63 species were up-regulated and 14 species were down-regulated, and 71 species of new lncRNA110 were up-regulated and 39 species down-regulated. There were 437 species of mRNA with differentially expressed mRNA in all the samples.Among them, 365 species were highly expressed and 72 were low expression.3)緇弎RT-PCR楠岃瘉闀塊摼闈炵紪鐮丷NA NONHSAT250987, NONHSAT176469,NONHSAT309646,NONHSAT140585, NONHSAT201926琛ㄨ揪鍛堜笅璋冭秼鍔，

本文編號：1707656