变应性鼻炎相关的长链非编码RNA表达谱分析

发布时间：2018-04-04 00:53

  本文选题：变应性鼻炎　切入点：RNA-seq测序　出处：《中南大学》2014年硕士论文

【摘要】：目的 探讨长链非编码RNA (Long Noncoding RNA, lncRNA)在变应性鼻炎与正常鼻粘膜中是否存在表达差异,并筛选出差异显著的长链非编码RNA。 方法 1)分别提取变应性鼻炎和正常鼻粘膜组织总RNA,质量检测后合成双链cDNA, PCR扩增建立cDNA文库,用Illumina HiSeqTM2000测序得到原始序列数据。运用软件Tophat、Cufflinks构建转录本。运用blast与non-coding RNA、kegg、nr、cog、swissprot等数据库比对,识别出已知的lncRNA和：mRNA;利用CPC基于framefinder和blast进行SVM预测,得到预测的新的非编码RNA。 2)运用RPKM法分别计算lncRNA和mRNA表达量,基于差异倍数运用聚类分析筛选出多对样本均有差异表达的lncRNA和mRNA。 3) qRT-PCR对部分差异表达的长链非编码RNA进行验证。 4)运用关系式数据分析系统RDAS计算差异表达的lncRNA和所有mRNA的关联性,得到与差异表达的lncRNA共表达的mRNA。 5)运用数据库KEGG、BioCarta对共表达的mRNA进行Pathway分析,得到共表达的mRNA参与的生物学途径。结合生物学途径结果和关联性结果了解lncRNA的调控作用。 结果 1)本研究首次应用RNA-Seq测序技术得到变应性鼻炎和正常鼻粘膜的lncRNA表达谱和mRNA表达谱。 2)3对样本中均有差异表达的lncRNA共173种,120种上调,53种下调；已知的lncRNA63种,49种上调,14种下调；新的lncRNA110个,71种上调,39种下调。3对样本中均有差异表达的mRNA共437种,其中365种高表达,72种低表达。 3)经qRT-PCR验证长链非编码RNA NONHSAT250987, NONHSAT176469、NONHSAT309646、NONHSAT140585、 NONHSAT201926表达呈下调趋势；长链非编码RNA NONHSAT26156、NONHSAT292828、NONHSAT312751、 NONHSAT157109表达呈上调趋势；结果与RNA-seq预测结果相一致。 4)关联性分析得到与85种差异表达的lncRNA共表达的mRNA,共379种。 5)经Pathway分析得到共表达的mRNA的17条生物学途径。 结论 变应性鼻炎患者鼻粘膜lncRNA的表达与正常鼻粘膜比较存在显著性差异。
[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)缁弎RT-PCR楠岃瘉闀块摼闈炵紪鐮丷NA NONHSAT250987, NONHSAT176469,NONHSAT309646,NONHSAT140585, NONHSAT201926琛ㄨ揪鍛堜笅璋冭秼鍔，

本文编号：1707656