In situ hybridization assay for circular RNA visualization based on padlock probe and rolling circle amplification

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Circular RNAs (circRNAs) are a class of noncoding RNAs generated through backsplicing.
• Thousands of circRNAs have been identified, but their functions remain largely unknown.
• In situ hybridization techniques are crucial for understanding circRNA localization and function.

Purpose of the Study:

• To develop a simple and effective method for in situ detection of circRNAs.
• To evaluate the performance of the new method against a commercial assay.

Main Methods:

• Developed a novel in situ detection method utilizing padlock probe hybridization and rolling circle amplification (RCA).
• Applied the method for detecting the circRNA Cdr1as in mouse brain tissue.
• Compared the detection efficiency with the commercially available BaseScope assay.

Main Results:

• The developed padlock probe assay successfully detected circRNAs in situ.
• The method demonstrated comparable detection efficiency to the BaseScope assay.
• The assay provides a straightforward and alternative approach for circRNA research.

Conclusions:

• The padlock probe hybridization and RCA method is a viable and simple tool for in situ circRNA detection.
• This technique can aid researchers in elucidating the biological and physiological functions of circRNAs.
• It offers a valuable alternative for the research community studying circRNA localization and dynamics.