Analysis of transcript and protein overlap in a human osteosarcoma cell line
Analysis of transcript and protein overlap in a human osteosarcoma cell line
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DOI：
10.1186/1471-2164-11-684
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发表时间：
2010-12-02
期刊：
影响因子：
4.4
通讯作者：
Lundeberg, Joakim
中科院分区：
文献类型：
--
作者：
Klevebring, Daniel;Fagerberg, Linn;Lundeberg, Joakim
背景：基因组学和蛋白质组学的一个有趣的研究领域是比较转录组和蛋白质组之间的重叠。最近，在全基因组水平上分析基因和蛋白质表达的工具已经得到改进，包括新一代测序仪器的可用性和高通量基于抗体的方法来分析蛋白质的存在和定位。在这项研究中，我们使用大规模转录组测序(RNA-seq)研究了人骨肉瘤细胞系的转录组，并将其表达水平与基于抗体的免疫组织化学(IHC)和免疫荧光显微镜(IF)获得的原位蛋白质数据进行了比较。结果：基于2749个基因进行了大规模分析，对应于人类基因组中约13%的蛋白质编码基因。我们发现很大一部分被分析的基因同时存在RNA和蛋白质，这三种方法都检测到了60%的被分析的人类基因。仅有34个基因(1.2%)在转录或蛋白质水平上未被检测到。我们的数据表明，大多数人类基因在该细胞系中以可检测到的转录本或蛋白质水平表达。由于抗体的可靠性取决于可能的交叉反应，我们使用基于不同标准的不同可靠性分数的抗体来比较RNA和蛋白质数据，包括蛋白质印迹分析。3个平台检测到的基因产物一般都有较好的抗体验证分数，而仅通过抗体检测而不是通过RNA测序检测的基因产物通常含有更多的低分抗体。结论：这表明一些抗体以非特异性的方式染色细胞，用RNA-SEQ评估转录本的存在可以为相应抗体的验证提供指导。
Background: An interesting field of research in genomics and proteomics is to compare the overlap between the transcriptome and the proteome. Recently, the tools to analyse gene and protein expression on a whole-genome scale have been improved, including the availability of the new generation sequencing instruments and high-throughput antibody-based methods to analyze the presence and localization of proteins. In this study, we used massive transcriptome sequencing (RNA-seq) to investigate the transcriptome of a human osteosarcoma cell line and compared the expression levels with in situ protein data obtained in-situ from antibody-based immunohistochemistry (IHC) and immunofluorescence microscopy (IF).Results: A large-scale analysis based on 2749 genes was performed, corresponding to approximately 13% of the protein coding genes in the human genome. We found the presence of both RNA and proteins to a large fraction of the analyzed genes with 60% of the analyzed human genes detected by all three methods. Only 34 genes (1.2%) were not detected on the transcriptional or protein level with any method. Our data suggest that the majority of the human genes are expressed at detectable transcript or protein levels in this cell line. Since the reliability of antibodies depends on possible cross-reactivity, we compared the RNA and protein data using antibodies with different reliability scores based on various criteria, including Western blot analysis. Gene products detected in all three platforms generally have good antibody validation scores, while those detected only by antibodies, but not by RNA sequencing, generally consist of more low-scoring antibodies.Conclusion: This suggests that some antibodies are staining the cells in an unspecific manner, and that assessment of transcript presence by RNA-seq can provide guidance for validation of the corresponding antibodies.