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Multiplexed Analysis of Diverse RNA Classes via Hybridization Chain Reaction


Acharya, Aneesh (2016) Multiplexed Analysis of Diverse RNA Classes via Hybridization Chain Reaction. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z95M63N0.


Gene circuits are complex biological networks composed of numerous regulatory elements, including transcription factors, mRNAs, and microRNAs. Fluorescent in situ hybridization (FISH) is a powerful method for spatially mapping expression levels of RNA elements within an intact organism, but traditional methods exhibit at least one of the following drawbacks: low signal-to-background, arduous and/or destructive multiplexing, and non-quantitative signal. These issues are all overcome using in situ amplification based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger the self-assembly of fluorophore-labeled nucleic acid hairpins into tethered fluorescent amplification polymers. In situ HCR enables straightforward multiplexing, high signal-to-background, and quantitative signal. Here, we address three key scenarios in which HCR enables novel applications for in situ hybridization. First, we address the challenge of sorting cell subpopulations based on mRNA abundance using flow cytometry to enable high-throughput measurement of the signal intensity from individual cells. High signal is required to overcome the background autofluorescence integrated over the volume of each cell. Quantitative HCR signal amplification enables multi-dimensional sorting of mammalian cell lines based on expression levels of multiple target mRNAs. Second, we address the challenge of mapping multiple microRNA and mRNA targets simultaneously. Traditional methods enable mapping of single microRNA targets in isolation and use costly LNA probes with proprietary compositions that differ for each target. Here we develop in situ HCR for multiplexed mapping not only of microRNAs, but of microRNAs and mRNAs together, using non-proprietary 2'OMe-RNA probes for miRNA targets and DNA probes for mRNA targets. Third, to enable studies of gut flora, we address the challenge of mapping spatial relationships between different bacterial species within the intact mouse colon. In situ HCR enables multiplexed discrimination of multiple closely-related Bacteroides species with rRNAs that differ by only a few nucleotides. In summary, this thesis presents in situ HCR as a tool for multiplexed analysis of diverse RNA classes and expands the range of gene circuit regulatory elements that can be spatially and quantitatively mapped.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:hybridization chain reaction; molecular programming; in situ hybridization; microRNA; bacteria mouse colon; flow cytometry; spectral imaging
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Bioengineering
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Pierce, Niles A.
Thesis Committee:
  • Elowitz, Michael B. (chair)
  • Pierce, Niles A.
  • Fraser, Scott E.
  • Mazmanian, Sarkis K.
Defense Date:19 May 2016
Non-Caltech Author Email:aneesh (AT)
Record Number:CaltechTHESIS:06032016-143422651
Persistent URL:
Acharya, Aneesh0000-0002-4402-7147
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9835
Deposited By: Aneesh Acharya
Deposited On:06 Jun 2016 20:21
Last Modified:08 Nov 2023 00:43

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