New tool isolates RNA within specific cells

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New tool isolates RNA within specific cells

University of Oregon biologists provide quick, precise view of cell development

and gene expression

http://www.eurekalert.org/pub_releases/2009-05/uoo-nti051709.php

A team of University of Oregon biologists, using fruit flies, has created a way

to isolate RNA from specific cells, opening a new window on how gene expression

drives normal development and disease-causing breakdowns.

While DNA (deoxyribonucleic acid) provides an identical genetic blueprint in

every cell, RNA (ribonucleic acid) decodes genetic instructions that turn

protein molecules on and off in different cell types.

The new tagging method, tested in a variety of subsets of Drosophila brain

cells, is described in a paper put on line ahead of regular publication by the

journal Nature Methods. Instead of scientists needing to physically separate

cell types, they now can inject a chemically modified gene from the one-celled

organism Toxoplasma gondii and activate it in only one cell type within a

tissue. Only newly generated RNA in this cell type is then tagged and isolated.

" By analyzing RNA from different cell types, we can begin to understand how

cellular differences are generated, " said lead author R. , a

National Science Foundation-funded doctoral student in the lab of Doe, a

UO biologist and Medical Institute (HHMI) investigator. " Our new

TU-tagging method should be useful for isolating cell-type specific RNA from

other organisms, including mammals, and should be useful in broad areas of

research including studies of development, neurobiology and disease. "

The new non-toxic, non-invasive method makes it possible to " listen in " to the

messages -- in fact, messenger RNA -- that the nucleus is sending each cell,

without perturbing the cell, Doe said. " It is much like eavesdropping on a phone

conversation, rather than pulling the person out of the house for questioning.

The cell has no idea that its RNAs are being 'tagged' for isolation and study.

That's good, because we get a more accurate idea of what the cell is saying. "

That, Doe added, could be helpful for 'listening' to host cells before and after

the initiation of a disease to determine how cells respond, or, for example

study healthy immune cells versus bacterially-challenged immune cells or neurons

before they learn a task and after they learn a task to determine what changes

in the cell are caused by the experience.

The new UO-developed tool builds on work led by co-author D. Cleary, who

as a doctoral student at Stanford University unveiled the T. gondii-based

approach for use in analyzing RNA synthesis and decay in 2005 in Nature

Biotechnology. Cleary, now a faculty member at the University of California,

Merced, worked on the UO project as a postdoctoral fellowship funded by the

National Institutes of Health and HHMI.

Cleary's group built its tool with the enzyme uracil phosphoribosyltransferase

(UPRT), a nucleotide salvage enzyme that prepares nucleotides for incorporation

into newly synthesized RNA. By altering the nucleotide analog 4-thiouracil, the

UPRT enzyme caused RNA to become tagged with thiouracil (TU), allowing the

" TU-tagged " RNA to be purified from untagged RNA.

In Doe's lab, , Cleary and research technician J. of the

UO's institutes of Neuroscience and Molecular Biology manipulated Drosophila so

that they would only express UPRT in specific target cells. The group tested the

new approach in embryos, larvae and adults using microarray technology to detect

cell type-specific gene expression. The researchers say the method should work

in other systems, including vertebrates, by using gene transfer, retroviral

delivery, electrical pulses of molecules through cell membranes, or messenger

RNA injection.