Understanding diverse RNA function using quantum dot based fluorescence in situ hybridization
Ribonucleic acids (RNAs) serve as one of the main building blocks of organic life and with the advent of new understanding beyond an intermediary for protein coding, understanding it's function becomes imperative. Previous tools study RNA expression from cell populations such as tissue or cell lines, however, an expansive area of research has developed for the individual cells. The recognition of cell heterogeneity in normal and diseased tissues further highlighted the importance of accurate RNA detection in single cells. Single molecule RNA fluorescence in situ hybridization (smRNA-FISH) has revolutionized RNA detection and quantification by visualizing single RNA transcripts.
The work in this dissertation improves upon smRNA-FISH by developing quantum dot based fluorescent in situ technologies that uses hybridization of quantum dot-labeled single stranded DNA oligonucleotides to the RNA targets for visualization and quantification. In chapter 2, we demonstrate our proof-of-concept quantum dot single molecule RNA-FISH (qdot-smRNA-FISH) by comparing the sensitivity and specificity of qdot-smRNA-FISH against commercialized smRNA-FISH. In chapter 3, we describe qdot-smRNA-FISH for detection of isoforms (isoFISH), where different from smFISH that requires dozens of hybridization probes per gene, isoFISH requires only two hybridization probes per splicing variant. In chapter 4, we used the co-localization of multi-color quantum dot FISH (CoQ-FISH) to observe for the first time a fusion transcript formation of Eml4-Alk in non-small cell lung cancer biopsy samples from trans-splicing and not chromosomal rearrangement. In chapter 5, we develop cell surface qDot RNA-FISH (Surface-FISH), a method for visualization of RNAs on the cell surface. We identified RNA targets using RNA-seq on membrane coated nanoparticles from EL4 and applied Surface-FISH to observe regions of Malat1 and Neat1. In chapter 6, we developed Surface-PromFISH that uses randomized antisense probes to promiscuously label as many csRNAs as possible and made it compatible with co-staining of cell membrane proteins. We subjected the hybridization targets to RNA sequencing (Surface-FISHseq), which led to 51 putative PBMC csRNAs and we identified a potential function where antisense blocking of FNDC3B and CTSS lowers monocyte attachment.