Details

Autor(en) / Beteiligte

• Xiao, Wen
• Yeom, Kyu-Hyeon
• Lin, Chia-Ho
• Black, Douglas L

Titel

Improved enzymatic labeling of fluorescent in situ hybridization probes applied to the visualization of retained introns in cells

Ist Teil von

• RNA (Cambridge), 2023-08, Vol.29 (8), p.1274-1287

Ort / Verlag

United States: Cold Spring Harbor Laboratory Press

Erscheinungsjahr

2023

Quelle

MEDLINE

Beschreibungen/Notizen

• Fluorescence in situ hybridization (FISH) is a widely used tool for quantifying gene expression and determining the location of RNA molecules in cells. We present an improved method for FISH probe production that yields high-purity probes with a wide range of fluorophores using standard laboratory equipment at low cost. The method modifies an earlier protocol that uses terminal deoxynucleotidyl transferase to add fluorescently labeled nucleotides to synthetic deoxyoligonucleotides. In our protocol, amino-11-ddUTP is joined to an oligonucleotide pool prior to its conjugation to a fluorescent dye, thereby generating pools of probes ready for a variety of modifications. This order of reaction steps allows for high labeling efficiencies regardless of the GC content or terminal base of the oligonucleotides. The degree of labeling (DOL) for spectrally distinct fluorophores (Quasar, ATTO, and Alexa dyes) was mostly >90%, comparable with commercial probes. The ease and low cost of production allowed the generation of probe sets targeting a wide variety of RNA molecules. Using these probes, FISH assays in C2C12 cells showed the expected subcellular localization of mRNAs and pre-mRNAs for (RNA polymerase II subunit 2a) and , and of the long noncoding RNAs and Developing FISH probe sets for several transcripts containing retained introns, we found that retained introns in the and transcripts are present in subnuclear foci separate from their sites of synthesis and partially coincident with nuclear speckles. This labeling protocol should have many applications in RNA biology.