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- All Subjects: Gene Expression
- Creators: Meldrum, Deirdre R.
Description
This thesis research focuses on developing a single-cell gene expression analysis method for marine diatom Thalassiosira pseudonana and constructing a chip level tool to realize the single cell RT-qPCR analysis. This chip will serve as a conceptual foundation for future deployable ocean monitoring systems. T. pseudonana, which is a common surface water microorganism, was detected in the deep ocean as confirmed by phylogenetic and microbial community functional studies. Six-fold copy number differences between 23S rRNA and 23S rDNA were observed by RT-qPCR, demonstrating the moderate functional activity of detected photosynthetic microbes in the deep ocean including T. pseudonana. Because of the ubiquity of T. pseudonana, it is a good candidate for an early warning system for ocean environmental perturbation monitoring. This early warning system will depend on identifying outlier gene expression at the single-cell level. An early warning system based on single-cell analysis is expected to detect environmental perturbations earlier than population level analysis which can only be observed after a whole community has reacted. Preliminary work using tube-based, two-step RT-qPCR revealed for the first time, gene expression heterogeneity of T. pseudonana under different nutrient conditions. Heterogeneity was revealed by different gene expression activity for individual cells under the same conditions. This single cell analysis showed a skewed, lognormal distribution and helped to find outlier cells. The results indicate that the geometric average becomes more important and representative of the whole population than the arithmetic average. This is in contrast with population level analysis which is limited to arithmetic averages only and highlights the value of single cell analysis. In order to develop a deployable sensor in the ocean, a chip level device was constructed. The chip contains surface-adhering droplets, defined by hydrophilic patterning, that serve as real-time PCR reaction chambers when they are immersed in oil. The chip had demonstrated sensitivities at the single cell level for both DNA and RNA. The successful rate of these chip-based reactions was around 85%. The sensitivity of the chip was equivalent to published microfluidic devices with complicated designs and protocols, but the production process of the chip was simple and the materials were all easily accessible in conventional environmental and/or biology laboratories. On-chip tests provided heterogeneity information about the whole population and were validated by comparing with conventional tube based methods and by p-values analysis. The power of chip-based single-cell analyses were mainly between 65-90% which were acceptable and can be further increased by higher throughput devices. With this chip and single-cell analysis approaches, a new paradigm for robust early warning systems of ocean environmental perturbation is possible.
ContributorsShi, Xu (Author) / Meldrum, Deirdre R. (Thesis advisor) / Zhang, Weiwen (Committee member) / Chao, Shih-hui (Committee member) / Westerhoff, Paul (Committee member) / Arizona State University (Publisher)
Created2013
Description
A major goal of the Center for Biosignatures Discovery Automation (CBDA) is to design a diagnostic tool that detects novel cancer biosignatures at the single-cell level. We designed the Single-cell QUantitative In situ Reverse Transcription-Polymerase Chain Reaction (SQUIRT-PCR) system by combining a two-photon laser lysis (2PLL) system with a microfluidic reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) platform. It is important to identify early molecular changes from intact tissues as prognosis for premalignant conditions and develop new molecular targets for prevention of cancer progression and improved therapies. This project analyzes RNA expression at the single-cell level and presents itself with two major challenges: (1) detecting low levels of RNA and (2) minimizing RNA absorption in the polydimethylsiloxane (PDMS) microfluidic channel. The first challenge was overcome by successfully detecting picogram (pg) levels of RNA using the Fluidigm (FD) BioMark™ HD System (Fluidigm Corporation, South San Francisco, CA) for RT-qPCR analysis. This technology incorporates a highly precise integrated fluidic circuit (IFC) that allows for high-throughput genetic screening using microarrays. The second challenge entailed collecting data from RNA flow-through samples that were passed through microfluidic channels. One channel was treated with a coating of polyethylene glycol (PEG) and the other remained untreated. Various flow-through samples were subjected to RT-qPCR and analyzed using the FD FLEXsix™ Gene Expression IFC. As predicted, the results showed that the treated PDMS channel absorbed less RNA than the untreated PDMS channel. Once the optimization of the PDMS microfluidic platform is complete, it will be implemented into the 2PLL system. This novel technology will be able to identify cell populations in situ and could have a large impact on cancer diagnostics.
ContributorsBlatt, Amy Elissa (Author) / Meldrum, Deirdre R. (Thesis director) / Tran, Thai (Committee member) / Chao, Joseph (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2014-05
Description
This thesis research focuses on phylogenetic and functional studies of microbial communities in deep-sea water, an untapped reservoir of high metabolic and genetic diversity of microorganisms. The presence of photosynthetic cyanobacteria and diatoms is an interesting and unexpected discovery during a 16S ribosomal rRNA-based community structure analyses for microbial communities in the deep-sea water of the Pacific Ocean. Both RT-PCR and qRT-PCR approaches were employed to detect expression of the genes involved in photosynthesis of photoautotrophic organisms. Positive results were obtained and further proved the functional activity of these detected photosynthetic microbes in the deep-sea. Metagenomic and metatranscriptomic data was obtained, integrated, and analyzed from deep-sea microbial communities, including both prokaryotes and eukaryotes, from four different deep-sea sites ranging from the mesopelagic to the pelagic ocean. The RNA/DNA ratio was employed as an index to show the strength of metabolic activity of deep-sea microbes. These taxonomic and functional analyses of deep-sea microbial communities revealed a `defensive' life style of microbial communities living in the deep-sea water. Pseudoalteromonas sp.WG07 was subjected to transcriptomic analysis by application of RNA-Seq technology through the transcriptomic annotation using the genomes of closely related surface-water strain Pseudoalteromonas haloplanktis TAC125 and sediment strain Pseudoalteromonas sp. SM9913. The transcriptome survey and related functional analysis of WG07 revealed unique features different from TAC125 and SM9913 and provided clues as to how it adapted to its environmental niche. Also, a comparative transcriptomic analysis of WG07 revealed transcriptome changes between its exponential and stationary growing phases.
ContributorsWu, Jieying (Author) / Meldrum, Deirdre R. (Thesis advisor) / Zhang, Weiwen (Committee member) / Abbaszadegan, Morteza (Committee member) / Neuer, Susanne (Committee member) / Arizona State University (Publisher)
Created2013
Description
The regulation of gene expression, timing, location, and amount of a given project, ultimately affects the cellular structure and function. More broadly, gene regulation is the basis for cellular differentiation and development. However, gene expression is not uniform among individuals and varies greatly between genetic males and females. Males are hemizygous for the X chromosome, whereas females have two X chromosome copies. Contributing to the sex differences in gene expression between males and females are the sex chromosomes, X and Y. Gene expression differences on the autosomes and the X chromosome between males (46, XY) and females (46, XX) may help inform on the mechanisms of sex differences in human health and disease. For example, XX females are more likely to suffer from autoimmune diseases, and genetic XY males are more likely to develop cancer. Characterizing sex-specific gene expression among human tissues will help inform the molecular mechanisms driving sex differences in human health and disease. This dissertation covers a range of critical aspects in gene expression. In chapter 1, I will introduce a method to align RNA-Seq reads to a sex chromosome complement informed reference genome that considers the X and Y chromosomes' shared evolutionary history. Using this approach, I show that more genes are called as sex differentially expressed in several human adult tissues compared to a default reference alignment. In chapter 2, I characterize gene expression in an early formed tissue, the human placenta. The placenta is the DNA of the developing fetus and is typically XY male or XX female. There are well-documented sex differences in pregnancy complications, yet, surprisingly, there is no observable sex difference in expression of innate immune genes, suggesting expression of these genes is conserved. In chapter 3, I investigate gene expression in breast cancer cell lines. Cancer arises in part due to the disruption of gene expression. Here I show 19 tumor suppressor genes become upregulated in response to a synthetic protein treatment. In chapter 4, I discuss gene and allele-specific expression in Nasonia jewel wasp. Chapter 4 is a replication and extension study and discusses the importance of reproducibility.
ContributorsOlney, Kimberly (Author) / Wilson, Melissa A (Thesis advisor) / Hinde, Katherine (Committee member) / Buetow, Kenneth (Committee member) / Banovich, Nicholas (Committee member) / Arizona State University (Publisher)
Created2021