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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2017)

Though numerous treatments for breast cancer have been developed, recurrence still exists, biomarkers and risk assessment tools are thus desired by the field. Cell-free RNA in blood or serum is now receiving more and more interest as a pool of biomarkers and RNA sequencing was believed to be a powerful tool for its analysis. However, development of the field was hindered by large volume of serum required for RNA extraction.

In this study, by circumventing RNA extraction and with the inspiration from single-cell RNA sequencing, we developed a technology being able to construct RNA sequencing libraries in large scale with microliters of direct unprocessed serum input. Optimizations for sequencing and mapping qualities, library complexity and library construction efficiency were conducted by varying serum input volume, adding custom sequencing primer and applying liquid handling instrument epMotion 5075. The optimized strategy “construct 96 different libraries in one single automated batch with 7ul serum input” was proposed accordingly. A huge diversity of genes could be found in serum with this technology. The developed technology was then applied to 96 breast cancer patients’ serums for its performance and potential in clinical cases. Based on the sequencing data: 465, 601 and 1259 genes were identified by three methods respectively to behave differently in recurrence and non-recurrence patients; The two populations could be separated by principle component analysis with all three sets of genes mentioned above; Preliminary recurrence risk assessment was conducted successfully with the classifier random forest.

    Cover page: Cell-free RNA Sequencing from Microliters of Unprocessed Serum
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2024)
    Non-invasively evaluating gene expression products in human pre-implantation embryos remains a significant challenge. Here, we develop a non-invasive method for comprehensive characterization of the extracellular RNAs (exRNAs) in a single droplet of spent media that was used to culture human in vitro fertilization embryos. We generate the temporal extracellular transcriptome atlas (TETA) of human pre-implantation development. TETA consists of 245 exRNA sequencing datasets for five developmental stages. These data reveal approximately 4,000 exRNAs at each stage. The exRNAs of the developmentally arrested embryos are enriched with the genes involved in negative regulation of the cell cycle, revealing an exRNA signature of developmental arrest. Furthermore, a machine-learning model can approximate the morphology-based rating of embryo quality based on the exRNA levels. These data reveal the widespread presence of coding gene-derived exRNAs at every stage of human pre-implantation development, and these exRNAs provide rich information on the physiology of the embryo.
      Cover page: A temporal extracellular transcriptome atlas of human pre-implantation development.
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2020)
        Cover page: Reply to Verwilt et al.: Experimental evidence against DNA contamination in SILVER-seq
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2019)
        Extracellular RNAs (exRNAs) are present in human serum. It remains unclear to what extent these circulating exRNAs may reflect human physiologic and disease states. Here, we developed SILVER-seq (Small Input Liquid Volume Extracellular RNA Sequencing) to efficiently sequence both integral and fragmented exRNAs from a small droplet (5 μL to 7 μL) of liquid biopsy. We calibrated SILVER-seq in reference to other RNA sequencing methods based on milliliters of input serum and quantified droplet-to-droplet and donor-to-donor variations. We carried out SILVER-seq on more than 150 serum droplets from male and female donors ranging from 18 y to 48 y of age. SILVER-seq detected exRNAs from more than a quarter of the human genes, including small RNAs and fragments of mRNAs and long noncoding RNAs (lncRNAs). The detected exRNAs included those derived from genes with tissue (e.g., brain)-specific expression. The exRNA expression levels separated the male and female samples and were correlated with chronological age. Noncancer and breast cancer donors exhibited pronounced differences, whereas donors with or without cancer recurrence exhibited moderate differences in exRNA expression patterns. Even without using differentially expressed exRNAs as features, nearly all cancer and noncancer samples and a large portion of the recurrence and nonrecurrence samples could be correctly classified by exRNA expression values. These data suggest the potential of using exRNAs in a single droplet of serum for liquid biopsy-based diagnostics.
          Cover page: Extracellular RNA in a single droplet of human serum reflects physiologic and disease states
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