Dataset Information

Isolation of mRNA from fly photoreceptor cells by mRNA tagging

ABSTRACT: 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

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE1790 | GEO | 2004/10/11

SECONDARY ACCESSION(S): PRJNA90587

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

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.

Project description:To investigate the in vivo gene expression changes in Drosophila intestinal stem cells (ISCs) when OXPHOS is disrupted and the role of FOXO in regulating this process, we dissected midguts from 4 groups of adult flies: "esg-GAL4,tub-GAL80TS,UAS-nlsGFP/UAS-mitoXhoI;UAS-pAbp-FLAG/+ & homoplasmic mt:CoIsyn" (C group), "esg-GAL4,tub-GAL80TS,UAS-nlsGFP/UAS-mitoXhoI;UAS-pAbp-FLAG/+ & heteroplasmic mt:CoIT300I" (H group, also called M group), "esg-GAL4,tub-GAL80TS,UAS-nlsGFP/UAS-mitoXhoI;UAS-pAbp-FLAG/UAS-FOXO-IR & homoplasmic mt:CoIsyn" (FC group), "esg-GAL4,tub-GAL80TS,UAS-nlsGFP/UAS-mitoXhoI;UAS-pAbp-FLAG/UAS-FOXO-IR & heteroplasmic mt:CoIT300I" (FH group, also called FM group), ~200 midguts per group. The flies were initially obtained and maintained at 18 °C till 2 days after eclosion and were then heat-shocked at 37 ºC for 2 hours and maintained in 29 °C for 5 days. Thus, the ISCs of H and FH group flies were carrying the lethal homoplasmic mitoXhoI-resistant mt:CoIT300I mitochondrial DNAs (mtDNAs) with the wild-type mtDNAs eliminated by mitoXhoI and the OXPHOS disrupted. Meanwhile, the ISCs of C and FC group flies were still carrying wild-type-like but mitoXhoI-resistant mt:CoIsyn mtDNAs. Through the mRNA tagging method with the expressed FLAG-tagged poly(A)-binding protein (pAbp) and anti-FLAG affinity pull-down, We purified and compared mRNA transcriptome profiles (RNA-seq) of different ISCs groups in duplicates. We found about 10% of the mRNAs we detected were either up-regulated or down-regulated in the OXPHOS-disrupted (M group) ISCs compared with control (C group). The down-regulated pool included many genes involved in cell differentiation, proliferation, and growth regulation. The up-regulated pool was enriched in genes participating in various metabolic processes, including glycolysis and fatty acids synthesis. This result suggests a possible metabolic adaptation in response to the OXPHOS deficiency in M group ISCs. Importantly, knockdown of FOXO in OXPHOS-disrupted ISCs restored the expression level of more than 50% of the up- or down-regulated genes, indicating that transcriptional response to OXPHOS deficiency is partially mediated by FOXO. We also noticed that genes whose expressions were altered in OXPHOS-disrupted ISCs included many related to Notch signaling, indicating the mis-regulation of this signaling in these ISCs.

Dataset Information

Isolation of mRNA from fly photoreceptor cells by mRNA tagging

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets