A simple smiFISH pipeline to quantify mRNA at the single-cell level in 3D

Abstract

Summary: Techniques allowing the precise quantification of mRNA at the cellular level are essential for understanding biological processes. Here, we present a semi-automated smiFISH (single-molecule inexpensive FISH) pipeline enabling quantification of mRNA in a small number of cells (∼40) in fixed whole mount tissue. We describe steps for sample preparation, hybridization, image acquisition, cell segmentation, and mRNA quantification. Although the protocol was developed in Drosophila, it can be optimized for use in other organisms.For complete details on the use and execution of this protocol, please refer to Guan et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Bioinformatics
• Cell Biology
• Developmental biology
• Microscopy
• Molecular Biology
• In Situ Hybridization