Abstract
RNA tracking allows researchers to visualize RNA molecules in cells and tissues, providing important spatio-temporal information regarding RNA dynamics and function. Methods such as fluorescent in situ hybridization (FISH) and molecular beacons rely on complementary oligonucleotides to label and view endogenous transcripts. Other methods create artificial chimeric transcripts coupled with bacteriophage-derived coat proteins (e.g. MS2, λN) to tag molecules in live cells. In other approaches, endogenous RNAs are recognized by complementary RNAs complexed with noncatalytic Cas proteins. Each technique has its own set of strengths and limitations that must be considered when planning an experiment. Here, we discuss the mechanisms, advantages, and weaknesses of in situ hybridization, molecular beacons, MS2 tagging and Cas-derived systems, as well as how RNA tracking can be employed to study various aspects of molecular biology.
Highlights
RNA molecules have a broad range of roles in the cell
This review describes the most popular among these techniques; we discuss methodologies to tag endogenous RNAs by using fluorescent oligomer tags (fluorescent in situ hybridization (FISH)) or beacons, to track individual RNAs by making chimeric RNAs bearing tractable elements (e.g. MS2, boxB, RNA aptamers), and to identify endogenous RNAs through a complementary RNA complexed with noncatalytic Cas9/Cas13
While RNA visualization is commonly used in basic biomedical research, these techniques are not widely used in diagnostics, where reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) analysis is preferred for the detection of oncogenic or viral transcripts [38]
Summary
RNA molecules have a broad range of roles in the cell. Coding RNAs (messenger (m)RNAs) serve as templates for the translation of proteins, while noncoding RNAs (including microRNAs (miRNAs) and long non coding RNAs (lncRNAs)) regulate gene expression programmes on many levels [1,2]. Immunoprecipitation (IP) of native or cross-linked ribonucleoprotein (RNP) complexes (RIP and CLIP, respectively) can further identify the targets of a given RNA-binding protein and provide insight into RNA–protein interactions [8] These methods have transformed the RNA field. Antisense oligonucleotides (ASOs) with autoradiographic labels such as 3H or 32P allowed researchers to target DNA or RNA sequences that were complementary to the oligomer, allowing the visualization of these sequences inside fixed cells or tissues [21] Over the years, this technique evolved with the introduction of new methods of detection, such as gold labelling in conjunction with electron microscopy or enzyme-linked chromogenic reporters [21]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
