Uniquely quantifying highly similar RNA transcripts at the single molecule level

RNA;FISH;Fluorescenct in situ hybridization;Single molecule;Single cell;Yeast;C.elegans;Strand displacement

Wadsworth, Gable Marsh ; Kim, Harold D. Physics Ratcliff, Will Gumbart, James C. Yunker, Peter Wartell, Roger ; Kim, Harold D.

University:Georgia Institute of Technology

Department:Physics

关键词: RNA;    FISH;    Fluorescenct in situ hybridization;    Single molecule;    Single cell;    Yeast;    C.elegans;    Strand displacement;   

Others  :  https://smartech.gatech.edu/bitstream/1853/62219/1/WADSWORTH-DISSERTATION-2018.pdf

美国|英语

来源: SMARTech Repository

【 摘 要 】

Understanding RNA expression is critical to understand the connection between phenotype and genotype in living cells. To further complicate things, RNA splicing leads to the generation of multiple distinct isoforms which are developmentally regulated and highly homologous. Current methods for identifying transcripts in situ are unable to uniquely identify most isoforms due to the fact that the sequence is shared. While these splicing variations can be identified using ensemble methods such as RNA-seq, this does not preserve spatial information. We demonstrate new Fluorescence In Situ Hybridization (FISH) techniques based on a short probe and hybridization chain reaction, which can uniquely identify RNA isoforms in both budding yeast and C. Elegans. We establish that with a singly labeled FISH probe ~60% of transcripts can be quantified. We demonstrate this technique on the SUS1 gene in single yeast cells and on the NURF1 gene in the C. Elegans germline. This technique enables cost effective interrogation of all of the isoforms of an RNA transcript.