Format

Send to

Choose Destination
Curr Biol. 2009 Dec 15;19(23):2037-42. doi: 10.1016/j.cub.2009.10.028.

Visualization of individual Scr mRNAs during Drosophila embryogenesis yields evidence for transcriptional bursting.

Author information

1
Section of Cell and Developmental Biology, Division of Biology, University of California, San Diego, La Jolla, CA 92093, USA. apare@ucsd.edu

Abstract

The detection and counting of transcripts within single cells via fluorescent in situ hybridization (FISH) has allowed researchers to ask quantitative questions about gene expression at the level of individual cells. This method is often preferable to quantitative RT-PCR, because it does not necessitate destruction of the cells being probed and maintains spatial information that may be of interest. Until now, studies using FISH at single-molecule resolution have only been rigorously carried out in isolated cells (e.g., yeast cells or mammalian cell culture). Here, we describe the detection and counting of transcripts within single cells of fixed, whole-mount Drosophila embryos via a combination of FISH, immunohistochemistry, and image segmentation. Our method takes advantage of inexpensive, long RNA probes detected with antibodies, and we present novel evidence to show that we can robustly detect single mRNA molecules. We use this method to characterize transcription at the endogenous locus of the Hox gene Sex combs reduced (Scr), by comparing a stably expressing group of cells to a group that only transiently expresses the gene. Our data provide evidence for transcriptional bursting, as well for divergent "accumulation" and "maintenance" phases of gene activity at the Scr locus.

PMID:
19931455
PMCID:
PMC2805773
DOI:
10.1016/j.cub.2009.10.028
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center