RNA-seq研究方法与策略.pptVIP

RNA-seq研究方法与策略;‘DNA makes RNA makes protein’;Messenger RNA (mRNA) is a large family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression. A non-coding RNA (ncRNA) is a functional RNA molecule that is not translated into a protein.;;一个典型人类细胞的RNA含量;RNA的特点;基于丰度的mRNA分类;Transcriptome The transcriptome is the complete set of transcripts in a cell, and their quantity, for a specific developmental stage or physiological condition. To catalogue all species of transcript (mRNAs, ncRNAs); To determine the transcriptional structure of genes (their start sites, 5′ and 3′ ends, splicing patterns and other post transcriptional modifications); To quantify the changing expression levels of each transcript during development and under different conditions.;但通常我们使用的是RNA-seq的狭义概念，亦即mRNA-seq。;Trends in “Transcriptome and RNA-seq”;.;GENReports: Market Tech Analysis, Produced by Enal Razvi, Ph.D. ? 2014 ;We asked: What is your major application for RNA-Seq?;1、RNA在生命过程中具有重要地位； 2、对DNA预测基因的修正； 3、疾病、肿瘤研究中具有重要作用； 4、生物与环境的相互作用； 5、基因表达调控研究； ……;直接得到核酸序列信息，除了得到基因表达量的差异，更可以检测RNA的结构和结构变异。 开放性的转录组分析：无需参考基因组信息，无需设计探针，不但能检测已知基因还能够发现新的转录本。 在测序覆盖率足够大时能够检测到细胞中的低丰度转录本。 随着测序深度的增加可以获得更广的动态检测范围，能够同时鉴定和定量高丰度转录本和低丰度转录本。 价格相对便宜;Applications;;Figure 1 RNA-seq work flow. Schematic diagram of RNA-seq library construction. Total RNA is extracted from 300,000 cells to 3 million cells, and a small aliquot is used to measure the integrity of the RNA. rRNA is then depleted through one of several methods to enrich subpopulation of RNA molecules, such as mRNA or small RNA. mRNA is fragmented into a uniform size distribution and the fragment size can be monitored by RNA gel electrophoresis or Agilent Bioanalyzer. The cDNA is then built into a library. The size distribution pattern of the library can be checked by Agilent Bioanalyzer; this information is important for RNA-seq data analysis. Mapping programs align r