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Mapping mRNA and Protein Expression with High Signal-to-Background in Diverse Organisms


Husain, Naeem Shahab (2016) Mapping mRNA and Protein Expression with High Signal-to-Background in Diverse Organisms. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9DF6P73.


In situ hybridization (ISH) techniques allow for the study of the nucleic acid expression within whole biological samples. The quality of probes for ISH dictates how accurate and bright the signal is for the experiment; however, there is currently not a systematic way to determine what the best probe set would be. In response to this, we have developed a framework to optimize an ISH probe set to achieve the greatest signal-to-background ratio. As methods like ISH help obtain more information about biological processes, there is a growing desire to simultaneously analyze various targets within the same sample to examine these complex genetic interactions. To facilitate this, a novel amplification technique called hybridization chain reaction (HCR) has allowed for the in situ detection of multiple target mRNAs concurrently in zebrafish embryos. We have now expanded this technology further by adapting HCR amplification for ISH to other model organisms, particularly, whole mount Drosophila melanogaster embryos and formalin-fixed parafin-embedded human tissue sections. Beyond looking at mRNA, immunohistochemistry (IHC) provides another tool to understand biological systems by analyzing protein expression patterns. The ability to easily look at both mRNAs and proteins in the same sample offers significant advantages as each provides unique information, but current methods are technically difficult and labor intensive. In response, we have engineered a scheme to use HCR to amplify signal for IHC. We then used this advancement to develop a straightforward protocol using HCR amplification for simultaneous detection of multiple proteins and mRNAs with a high signal-to-background ratio.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:hybridization chain reaction, in situ hybridization, immunohistochemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Pierce, Niles A.
Thesis Committee:
  • Cai, Long (chair)
  • Pierce, Niles A.
  • Prober, David A.
  • Sternberg, Paul W.
Defense Date:16 December 2015
Additional Information:The Caltech Option "Biochemistry and Molecular Biophysics" is considered an Interdisciplinary Program. The author studied in both the Biology and Biological Engineering (BBE) and the Chemistry and Chemical Engineering (CCE) divisions to earn her degree. He is listed under CCE in the 2016 Commencement program.
Funding AgencyGrant Number
NSF Graduate FellowshipUNSPECIFIED
Record Number:CaltechTHESIS:02252016-123239637
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9585
Deposited By: Naeem Husain
Deposited On:08 Mar 2017 21:17
Last Modified:08 Nov 2023 00:43

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