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Isolation of mRNA from specific tissues of Drosophila by mRNA tagging.

ABSTRACT: 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.

SUBMITTER: Yang Z

PROVIDER: S-EPMC1243647 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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Isolation of mRNA from specific tissues of Drosophila by mRNA tagging.

Yang Zhiyong Z Edenberg Howard J HJ Davis Ronald L RL

Nucleic acids research 20051004 17

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 ti ...[more]

PMID: 16204451

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Project description:The method of mRNA tagging was recently developed to isolate mRNA population from muscle tissues in Caenohabditis elegans. This method utilizes a FLAG-tagged poly(A)-binding protein (PABP), which can bind to poly(A)-tail of eukaryotic mRNA. Thus, the mRNA population from specific tissue, in which FLAG- tagged PABP is expressed, can be co-immunoprecipitated with FLAG-tagged PABP using FLAG-specific antibody. To evaluate the applicability of this method to isolate mRNA from specific tissue in Drosophila, we generated Rh1-GLA4/P{UAS-dPABP-FLAG} flies which express a FLAG-tagged PABP in photoreceptor cells R1-R6. We then prepared 4 individual mRNA samples (subset 1: GSM30900-30903)) presumably from photoreceptor cells of these flies by mRNA tagging method. We also prepared 4 individual mRNA samples (subset 2: GSM30832-30835) from whole heads of the Rh1-GAL4/P{UAS-dPABP-FLAG} flies, as well as 6 individual mRNA samples (subset 3: GSM30824-30828, GSM30830) from whole heads of wild type Canton-S flies. The total 14 mRNA samples were used to synthesize target separately and each of the resultant target was used to hybridize with 1 Drosophila Genome Array DrosGenome1 Affymetrix GeneChip. The raw data of the 14 microarray were imported to dChip program (version 1.3). The arrays were normalized to a common baseline array that has the median overall brightness. The expression value for each probe set on each chip was then calculated as model-based expression index (MBEI) by Perfect Match-only model. By comparing the results of subset 1 and subset 2, we found most known photoreceptor-specific genes were indeed enriched by mRNA tagging. Through the comparison of the results of subset 2 and subset 3, we found the mRNAs of most genes known to be involved in fly visual function were underrepresented in subset 2, which could be due to squelching effect of GAL4. By combining the data of subset 1, 2 and 3, we were able to identify 11 new photoreceptor cell-enriched genes. Keywords: other

Project description:BackgroundBerries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip(R) Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions.ResultsOverall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and transport processes. Seeds, which supply essential resources for embryo development, showed higher mRNA abundance of genes encoding phenylpropanoid biosynthetic enzymes, seed storage proteins, and late embryogenesis abundant proteins. Water-deficit stress affected the mRNA abundance of 13% of the genes with differential expression patterns occurring mainly in the pulp and skin. In pulp and seed tissues transcript abundance in most functional categories declined in water-deficit stressed vines relative to well-watered vines with transcripts for storage proteins and novel (no-hit) functional assignments being over represented. In the skin of berries from water-deficit stressed vines, however, transcripts from several functional categories including general phenypropanoid and ethylene metabolism, pathogenesis-related responses, energy, and interaction with the environment were significantly over-represented.ConclusionThese results revealed novel insights into the tissue-specific expression mRNA expression patterns of an extensive repertoire of genes expressed in berry tissues. This work also establishes an extensive catalogue of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern tissue-specific expression patterns associated with tissue differentiation within berries. These results also confirmed that water-deficit stress has a profound effect on mRNA expression patterns particularly associated with the biosynthesis of aroma and color metabolites within skin and pulp tissues that ultimately impact wine quality.

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Isolation of mRNA from specific tissues of Drosophila by mRNA tagging.

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Isolation of mRNA from specific tissues of Drosophila by mRNA tagging.

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