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.
Methods in Molecular Biology (Clifton, N.J.)
|March 19, 2024
Summary
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.
