Unknown,Transcriptomics,Genomics,Proteomics

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TU-tagging: cell type specific RNA isolation from intact complex tissues

ABSTRACT: We show that the combination of spatially restricted uracil phosphoribosyltransferase (UPRT) expression with 4-thiouracil (4TU) delivery can be used to label and purify cell type specific RNA from intact complex tissues. This method is useful for isolating RNA from cell types that are difficult to isolate by dissection or dissociation methods and should work in any organism where UPRT can be spatially expressed, including mammals and other vertebrates. To confirm that thio-labeled RNA was from UPRT-expressing cells and demonstrate the utility of TU-tagging for cell type-specific RNA isolation, we purified TU-tagged and untagged RNA and compared them using microarrays. We chose to isolate RNA from larval glia, which are present in low number, are highly dispersed, and have a complex cell morphology, making them one of the most difficult cell types to isolate by dissection or dissociation methods. We used reversed polarity (repo)-GAL4 to drive expression of UAS-UPRT specifically in glial cells of the larval brain. We purified TU-tagged and untagged RNA from 72-96h larval brains, and hybridized them to custom Agilent microarrays. We performed two biological replicate experiments.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Michael Miller

PROVIDER: E-GEOD-15445 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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TU-tagging: cell type-specific RNA isolation from intact complex tissues.

Miller Michael R MR Robinson Kristin J KJ Cleary Michael D MD Doe Chris Q CQ

Nature methods 20090510 6

We found that the combination of spatially restricted uracil phosphoribosyltransferase (UPRT) expression with 4-thiouracil delivery can be used to label and purify cell type-specific RNA from intact complex tissues in Drosophila melanogaster. This method is useful for isolating RNA from cell types that are difficult to isolate by dissection or dissociation methods and should work in many organisms, including mammals and other vertebrates. ...[more]

PMID: 19430475

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Project description:Population based studies have established that androgen deficiency in males correlates with type 2 diabetes, visceral adiposity, and metabolic syndrome. Androgen therapy has been investigated as a possible treatment regime to combat these disorders. However, the molecular mechanism of androgen effects on these diseases still remain poorly understood. The zucker diabetic fatty (ZDF) rat, containing a mutation in the leptin receptor, is a well-investigated model of obesity and type 2 diabetes. Male rats are characterized as androgen deficient and spontaneously develop obese, hyperlipidemia, hyperglycemia and hyperinsulinemia. In this study, we used ZDF male rats as a model of metabolic syndrome to investigate the effects of testosterone administration on the development of the metabolic conditions. Methods: Male ZDF rats at six week of age were randomly divided into two groups and administered testosterone undecanoate(TU) or vehicle alone every three days for three weeks. After three weeks, overnight fasted blood glucose and insulin concentrations were significantly higher and glucose tolerance and insulin sensitivity were impaired in TU treated ZDF rats compared to vehicle controls. Moreover, increased serum triglycerides and VLDL were observed in TU treated rats. To further explore the observed metabolic changes in TU treated ZDF rats, whole-genome microarray analysis were performed on isolated liver mRNA. Results: Array analysis revealed that many hepatic lipogenic genes were increased in male ZDF rat livers treated with TU. Interestingly, SREBP-1c, a key transcriptional activator of lipogenic genes and PGC-1 , an activator of SREBP-1c were induced while small heterodimer partner, a transcriptional inhibitor of lipogenic genes was suppressed by TU treatment. Exploring signaling pathways for these effects, we observed that the hepatic activated forms of STAT3 and AMPK, two known inhibitors of hepatic lipogenesis, were decreased in TU treated rat. Moreover, we observed that DHT could block the induction of STAT3 and AMPK phosphorylation in treated primary human hepatocytes. Preliminarily, in the leptin receptor positive zucker diabetic lean male rats, we observed that TU treatment has an oppose effect on the hepatic lipogenic genes, suggesting that hepatic leptin signaling may influence androgen signaling. Further insight into the relationship between androgen deficiency and the leptin system may help improve treatment of the metabolic syndrome. Population based studies have established that androgen deficiency in males correlates with type 2 diabetes, visceral adiposity, and metabolic syndrome. Androgen therapy has been investigated as a possible treatment regime to combat these disorders. However, the molecular mechanism of androgen effects on these diseases still remain poorly understood. The zucker diabetic fatty (ZDF) rat, containing a mutation in the leptin receptor, is a well-investigated model of obesity and type 2 diabetes. Male rats are characterized as androgen deficient and spontaneously develop obese, hyperlipidemia, hyperglycemia and hyperinsulinemia. In this study, we used ZDF male rats as a model of metabolic syndrome to investigate the effects of testosterone administration on the development of the metabolic conditions. Two-condition experiment. (1) lean ZDF rats (control) vs. lean ZDF rats (testosterone treated). (2) obese ZDF rats (control) vs. obese ZDF rats (testosterone treated). Biological replicates: 4 control replicates, 4 treated replicates.

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TU-tagging: cell type specific RNA isolation from intact complex tissues

Publications

TU-tagging: cell type-specific RNA isolation from intact complex tissues.

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