Purchase this article with an account.
Jessica Heap, Marie-Audrey Ines Kautzmann, Nicolas Guillermo Bazan; Gene signatures of single photoreceptor cells (PRCs) using microfluidic technology. Invest. Ophthalmol. Vis. Sci. 2017;58(8):635.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Single-cell technologies reveal important heterogeneity within cell populations and provide insight into the molecular and biochemical dynamics within and among individual cells. Presently there remains limitations in applying these methods to irregularly shaped cells such as PRCs. Here we describe the successful isolation of single PRCs and their subsequent qPCR processing using microfluidic technology.
A cell suspension from dissociated wild type adult mouse retinas was prepared using 5 U/ml papain + 2mM EDTA + 2.7 mM L-Cysteine for 5 min at 37 C, followed by trituration. After confirming the presence of PRCs using bright field microscopy and determining optimal cell concentration and buoyancy, 6uL of the cell suspension was loaded in the C1 (Fluidigm) for single-cell isolation. Successful capture of single PRCs, as well as some cell aggregates and outer segments, was observed using fluorescence from Hoechst and CalceinAM/Ethidium homodimer. The chip was then reloaded in the C1, which automatically performed cell lysis, reverse transcription of cell content and preamplification using retinal cell-specific primer pairs. The subsequent cDNA was loaded using the Juno (Fluidigm), followed by qPCR in the Biomark (Fluidigm).
Expression of several known PRC biomarkers including Rhodopsin, Peripherin2, and S-Opsin confirmed that the majority of our cell captures consisted of rod PRCs, with only a few cone PRCs. Furthermore, no expression of non-PRC retinal biomarkers such as Syntaxin1A, Calbindin, RLBP1 and PKC-alpha was detected. Principal component analysis and clustered heat maps revealed the presence of multiple unique gene expression profiles among the PRCs. In contrast, the cell aggregates showed high expression of the same PRC biomarkers, yet share a single gene expression profile.
We determined that a higher concentration of cDNA harvested from the C1 is required for optimal qPCR, however we demonstrate a successful method for single PRC capture and analysis using microfluidic technology. Importantly, our downstream analysis reveals unique gene signatures of single PRCs and shows how these expression profiles are lost in bulk cell analysis. Further investigation of the single PRC transcriptome is needed in order to fully understand the intra- and inter-cellular mechanisms and pathways which maintain homeostasis in these complex cells.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only