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

Area of Science:

• Molecular Biology
• Cell Biology
• Genomics

Background:

• Accurate mRNA quantification at the cellular level is crucial for understanding gene expression and biological mechanisms.
• Existing methods may lack efficiency or scalability for analyzing small cell populations in complex tissues.

Purpose of the Study:

• To develop and present a semi-automated pipeline for precise mRNA quantification in small cell numbers.
• To enable detailed analysis of gene expression within specific cellular contexts using single-molecule FISH (smiFISH).

Main Methods:

• The study details a protocol for sample preparation, hybridization, and image acquisition for fixed whole mount tissues.
• It includes steps for automated cell segmentation and subsequent mRNA spot counting for quantification.
• The pipeline, termed single-molecule inexpensive FISH (smiFISH), is semi-automated.

Main Results:

• The developed smiFISH pipeline allows for the precise quantification of mRNA molecules in approximately 40 cells within fixed whole mount tissues.
• The protocol is demonstrated in Drosophila but is designed for adaptability to other model organisms.

Conclusions:

• The semi-automated smiFISH pipeline offers an efficient method for cellular mRNA quantification in small tissue samples.
• This technique facilitates deeper insights into biological processes by enabling precise gene expression analysis at the single-cell level.