Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Jun;6(6):439-41.
doi: 10.1038/nmeth.1329. Epub 2009 May 10.

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

Michael R Miller  1 , Kristin J Robinson, Michael D Cleary, Chris Q Doe
Affiliations

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

Michael R Miller et al. Nat Methods. 2009 Jun.

Abstract

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1. TU-tagging: overview and cell type specific labeling
(A) TU-tagging procedure. (B) TU-tagging in larvae, and adults. RNA from larvae expressing GAL4 in no cell type (−), all neurons (elav), muscle cells (c179) or the mushroom body (OK 107) and either with (+) or without (−) UAS-UPRT, and adults of the indicated genotypes were electrophoresed and stained with ethidium bromide to detect all RNA (top) and streptavidinhorseradish peroxidase to detect thio-RNA (bottom). (C) TU-tagging in embryos. 0−16h embryos of the indicated genotypes were treated with 4TU for 2 hours. RNA was analyzed as indicated above. (D). Comparison of RNA labeling before and after tissue isolation. wor, neuroblasts.
Figure 2
Figure 2. Cell-type specific RNA isolation and analysis
(A) Single confocal section through a brain lobe from a 96h ALH larvae expressing HA:UPRT in all glia (repo-GAL4 UAS-HA:UPRT) stained for HA:UPRT (detected with an HA antibody) and glial nuclei (Repo). Scale bar, 20 μM.(B) fold-enrichment of selected larval glia-specific genes. See supplemental table 1 for all enriched genes. (C) Average microarray ratios from two glia TU-tagging experiments plotted against transcript uracil number. Dashed red line indicates cutoff for top 5% enriched genes. Green dots, previously known larval glia-specific genes; vertical arrowhead, possible false positives; horizontal arrowhead, possible false negatives. (D) TU-tagging microarray ratios after normalization and removal of transcripts with missing UTR annotations (see methods). (E) Comparison of top 1% and 5% enriched genes before and after normalization.
See this image and copyright information in PMC

References

    1. Tomancak P, Berman BP, Beaton A, et al. Genome Biol. 2007;8(7):R145. - PMC - PubMed
    1. Wang Z, Gerstein M, Snyder M. Nat Rev Genet. 2009;10(1):57. - PMC - PubMed
    1. Schadt EE, Monks SA, Friend SH. Biochem Soc Trans. 2003;31(2):437. - PubMed
    1. Roy PJ, Stuart JM, Lund J, et al. Nature. 2002;418(6901):975. - PubMed
    1. Tanke HJ, van der Keur M. Trends Biotechnol. 1993;11(2):55. - PubMed

Publication types

MeSH terms