hcHCR: High-Throughput Single-Cell Imaging of RNA in Human Primary Immune Cells
Manasi Gadkari1, Jing Sun2, Adrian Carcamo3
1Functional Immunogenomics Section, NIAMS/NIH, Bethesda, MD, USA.
Abstract:
Functional genomics and chemical screens can identify and characterize novel cellular factors regulating signaling networks and chemical tools to modulate their function for the treatment of disease. Screening methods have relied primarily on immortalized and/or transformed cancer cell lines, which can limit the generalization of results to more physiologically relevant systems. Most have also relied on immunofluorescence, or on stably expressed recombinant fluorescent proteins, to detect specific protein markers using high-content imaging readouts. In comparison, high-throughput methods to visualize and measure RNA species have been less explored. To address this, we have adapted an isothermal signal amplification chemistry for RNA FISH known as hybridization chain reaction (HCR) to an automated, high-content imaging assay format. We present a detailed protocol for this technique, which we have named high-content HCR (hcHCR). The protocol focuses on the measurement of changes in mRNA abundance at the single-cell level in human primary cells, but it can be applied to a variety of primary cell types and perturbing agents. We anticipate that hcHCR will be most suitable for low- to medium-throughput screening experiments in which changes in transcript abundance are the desired output measure.
Insights
We developed high-content hybridization chain reaction (HCR) for automated imaging, enabling single-cell mRNA measurement in primary cells. This method advances functional genomics screening beyond cancer cell lines.
Area of Science:
- Cellular and Molecular Biology
- Genomics and Proteomics
- Biotechnology and Bioengineering
Background:
- Functional genomics and chemical screens identify cellular factors and therapeutic targets.
- Current screening methods often use cancer cell lines and protein markers, limiting physiological relevance.
- High-throughput RNA visualization methods are less explored compared to protein detection.
Purpose of the Study:
- To adapt hybridization chain reaction (HCR) for automated, high-content imaging.
- To enable single-cell mRNA abundance measurement in human primary cells.
- To provide a protocol for a new screening technique called high-content HCR (hcHCR).
Main Methods:
- Adapted isothermal signal amplification chemistry (HCR) for RNA FISH.
- Developed an automated, high-content imaging assay format.
- Focused on measuring mRNA abundance changes at the single-cell level.
Main Results:
- Presented a detailed protocol for high-content HCR (hcHCR).
- Demonstrated application in human primary cells, adaptable to various cell types.
- Established hcHCR for measuring transcript abundance changes.
Conclusions:
- High-content HCR (hcHCR) offers a novel approach for RNA-based screening.
- The method allows for single-cell mRNA analysis in physiologically relevant primary cells.
- hcHCR is suitable for low- to medium-throughput screening focused on transcript abundance changes.
