Oligo(dT) Fluorescence In Situ Hybridization to Visualize the Poly(A) mRNAs in the Internal Tissues of Drosophila

Fluorescence in situ hybridization (FISH) is a cytological method used to visualize specific oligonucleotide sequences within the cell. This method relies on the specific binding of a fluorescence-tagged probe, a short stretch of single-stranded polynucleotide, to its complementary sequence in the DNA or RNA, forming stable double-stranded hybrids. Fluorochromes, such as fluorescein, Alexa Fluor, cyanine dyes, or rhodamine, are attached to these probes to help in detecting their presence within the cell. Based on sequence complementarity, FISH allows for the visualization of the DNA or RNA with which they have hybridized. The distribution of these fluorochrome-tagged probes can be observed under a fluorescence or confocal microscope. The oligo(dT) FISH technique specifically utilizes a fluorochrome-tagged stretch of 40-50 thymidine (T) oligonucleotides that binds to the poly(A) tails of mature mRNAs within the cell. Newly transcribing pre-mRNAs and certain non-coding RNAs may not have poly(A) tails and therefore cannot be detected by this method. This step-by-step protocol outlines the oligo(dT) FISH technique for visualizing the cellular distribution of polyadenylated mRNAs in the tissues of Drosophila and other related model organisms. Key features • Oligo (dT) fluorescence in situ hybridization is a robust cytological technique to visualize polyadenylated RNAs in the cell. • This oligo(dT) FISH protocol is applicable for a wide range of animals and cell culture model systems. • This protocol is particularly useful to visualize mRNAs in thin tissues with a few layers of cells.