RT Journal Article
SR Electronic
T1 Single-molecule FISH in Drosophila muscle reveals location dependent mRNA composition of megaRNPs
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 156091
DO 10.1101/156091
A1 Akiko Noma
A1 Carlas S. Smith
A1 Maximillian Huisman
A1 Robert M. Martin
A1 Melissa J. Moore
A1 David Grunwald
YR 2017
UL http://biorxiv.org/content/early/2017/06/26/156091.abstract
AB Single-molecule fluorescence in-situ hybridization (smFISH) provides direct access to the spatial relationship between nucleic acids and specific subcellular locations. The ability to precisely localize a messenger RNA can reveal key information about its regulation. Although smFISH is well established in cell culture or thin sections, methods for its accurate application to tissues are lacking. The utility of smFISH in thick tissue sections must overcome several challenges, including probe penetration of fixed tissue, accessibility of target mRNAs for probe hybridization, high fluorescent background, spherical aberration along the optical axis, and image segmentation of organelles. Here we describe how we overcame these obstacles to study mRNA localization in Drosophila larval muscle samples that approach 50 μm thickness. We use sample-specific optimization of smFISH, particle identification based on maximum likelihood testing, and 3-dimensional multiple-organelle segmentation. The latter allows using independent thresholds for different regions of interest within an image stack. Our approach therefore facilitates accurate measurement of mRNA location in thick tissues.