Yang Zhiyong, Edenberg Howard J, Davis Ronald L
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Nucleic Acids Res. 2005 Oct 4;33(17):e148. doi: 10.1093/nar/gni149.
To study the function of specific cells or tissues using genomic tools like microarray analyses, it is highly desirable to obtain mRNA from a homogeneous source. However, this is particularly challenging for small organisms, like Caenorhabditis elegans and Drosophila melanogaster. We have optimized and applied a new technique, mRNA tagging, to isolate mRNA from specific tissues of D.melanogaster. A FLAG-tagged poly(A)-binding protein (PABP) is expressed in a specific tissue and mRNA from that tissue is thus tagged by the recombinant PABP and separated from mRNA in other tissues by co-immunoprecipitation with a FLAG-tag specific antibody. The fractionated mRNA is then amplified and used as probe in microarray experiments. As a test system, we employed the procedures to identify genes expressed in Drosophila photoreceptor cells. We found that most known photoreceptor cell-specific mRNAs were identified by mRNA tagging. Furthermore, at least 11 novel genes have been identified as enriched in photoreceptor cells. mRNA tagging is a powerful general method for profiling gene expression in specific tissues and for identifying tissue-specific genes.
为了使用诸如微阵列分析等基因组工具来研究特定细胞或组织的功能，非常希望从同源来源获取mRNA。然而，对于像秀丽隐杆线虫和黑腹果蝇这样的小型生物体而言，这极具挑战性。我们优化并应用了一种新技术——mRNA标签法，从黑腹果蝇的特定组织中分离mRNA。一种带有FLAG标签的聚腺苷酸结合蛋白（PABP）在特定组织中表达，该组织的mRNA因此被重组PABP标记，并通过与FLAG标签特异性抗体的共免疫沉淀与其他组织中的mRNA分离。然后对分级分离的mRNA进行扩增，并用作微阵列实验的探针。作为一个测试系统，我们采用这些程序来鉴定在果蝇光感受器细胞中表达的基因。我们发现，大多数已知的光感受器细胞特异性mRNA通过mRNA标签法被鉴定出来。此外，至少有11个新基因被鉴定为在光感受器细胞中富集。mRNA标签法是一种强大的通用方法，可用于分析特定组织中的基因表达以及鉴定组织特异性基因。
