Isolation of mRNA from specific tissues of Drosophila by mRNA tagging.
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.
Yang
Zhiyong
Z
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Edenberg
Howard J
HJ
Davis
Ronald L
RL
D000818:Animals; D030801:Animals, Genetically Modified; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism; D039102:Poly(A)-Binding Proteins / Q000235:genetics; D015347:RNA Probes / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D005404:Fixatives; D009842:Oligopeptides; D010455:Peptides; D039102:Poly(A)-Binding Proteins; D015347:RNA Probes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005557:Formaldehyde; C085706:FLAG peptide