Research Department: Neurology and Neurotherapeutics Graduation Date: May 2019
Abstract: Single-cell RNA sequencing (scRNA-seq) gives researchers the ability to measure the distribution of gene expression within individual cells across a population. Complex tissues such, as the brain, contain many different functionally specialized cells. These cells use the same genome in different ways creating diverse cell types which respond to stimuli and alter their morphology due to mutations in unique ways. Before scRNA-seq, identifying cell-specific changes in the transcriptome was expensive, time-consuming and had a low throughput. This advancement opens the door to the identification of new cell types, and comparison of individual cell responses to certain stimuli. It also elucidates the dynamic nature of gene expression and the effects of gene regulatory networks across cell types. scRNA-seq differs from existing bulk tissue analyses and microarray techniques, in that it provides a unique expression profile for each input cell, as opposed to an average gene expression profile of a tissue. Single-cell genomic analysis can reveal variability among individual cells that may be crucial for maintaining normal functions in complex tissues and therefore is an invaluable approach in identifying the underlying mechanisms of disease.
One limitation of scRNA-seq is that it requires fresh tissue with intact cell membranes. An alternative to scRNA-seq is single nuclei RNA sequencing (sNuc-seq) which provides a similar ability to profile gene expression in a cell-specific manner by isolating nuclei as opposed to cells. This technique expands the input suspension to include frozen clinical samples, archived materials, tissues that cannot be dissociated and those from patients with neurodegenerative disorders. Additionally, it reduces the need for harsh enzymatic dissociation that harms the integrity of neuronal RNA and biases proportions of recovered cell types. Comparing the mRNA transcripts captured in scRNA-seq to those captured by sNuc-seq will also give us an understanding of localization of transcripts within the cell. Where the mRNA transcript localizes either in the cytosol or the nucleus may be an important factor in determining whether that transcript will be modified, translated, or degraded.
We aim to use both sNuc-seq and scRNA-seq techniques through the 10x Genomics Chromium Chip and Drop-Seq platforms in parallel to understand patterns in differential gene expression in frozen and fresh tissues and to compare the overall efficiency of the fluidic platforms and the bioinformatics pipelines.
What does research mean to you? Research gave me the opportunity to immerse myself in a topic of interest and pursue my curiosity. It has been a tremendous learning experience and will go on to shape not only the way I understand neuroscience and bioinformatics but also the way I approach challenges in other areas. The Green Fellowship gave me the chance to commit to a project independently and contribute to a much larger effort to understand the human brain and diseases that affect it.
Tell us about your journey. Put simply, I enjoy working at the Konopka Lab. As a freshman, I was inspired by the Green Fellowship posters and intended to apply to the program once I had more research experience. Nearly two years later, I found myself nervously choosing a lab for the Fellowship. I was overwhelmed by the opportunity at first, but my PI and lab members were extremely welcoming and helped me every step of the way. There was a steep learning curve during the first month but as the project progressed I took on more responsibility and began to expand my learning to new and exciting areas. I’m incredibly thankful to my lab and the Green fellowship administration for their support throughout the journey and for making the Green Fellowship my most formative college experience.
Advice for Future Green Fellows
Understand clearly what is expected from you early on. Don’t be afraid to ask questions and make mistakes, it’s part of the learning process. Attend seminars, poster sessions and take advantage of your time at the UTSW campus. Limit your commitments outside the fellowship. Plan to take the MCAT/GRE early. In my experience working full time and studying can be done but it is challenging. Regarding your poster, start early and practice tailoring your presentation to unique audiences.