Single-Cell Method Measures RNA and Proteins to Reveal Immune Responses

Single-cell RNA sequencing profiles transcriptional states, but proteins carry out those instructions and often do not mirror RNA, particularly for cytokines. This disconnect can obscure the timing and magnitude of immune signaling in individual cells. Researchers now describe a multimodal sequencing approach that measures RNA and proteins together to close these gaps.

Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine, with collaborators at the University of California, San Francisco and the Helen Diller Family Comprehensive Cancer Center, developed CIPHER-seq (Cytokine Intracellular Protein High-throughput Expression with RNA sequencing). The platform identifies cytokine activity more directly by capturing both transcriptional intent and protein output in the same immune cell. It provides a fuller view of immune responses that is aimed at clarifying how signaling unfolds at single-cell resolution.

CIPHER-seq works by gently preserving cells and simultaneously profiling multiple molecular layers. From a single immune cell, the method measures genome-wide RNA, cell-surface proteins, intracellular proteins, and cytokines that are retained inside the cell before secretion. In comparisons with standard approaches, CIPHER-seq induced far less cellular stress; cells processed by other methods showed signs of damage, particularly within mitochondria, which can confound downstream readouts.

To evaluate the platform, investigators activated immune cells and tracked responses over time. The assay detected increases in key cytokines, including interferon-gamma and tumor necrosis factor, and captured when these signals appeared. Using a computational trajectory to order cells along an activation timeline, RNA signals rose first and were followed by protein increases with a small but consistent delay, enabling stepwise tracking when both modalities are measured together.

The work is published in Scientific Reports on April 8, 2026. According to the team, integrating RNA with intracellular and surface protein measurements provides a stronger foundation for investigations of cancer biology, inflammatory processes, and mechanisms of treatment resistance across immune contexts.

“RNA gives us clues about where a cell is headed. Proteins show us where it actually arrives, and this clearer picture could help scientists design better immunotherapies and help clinicians predict which patients are most likely to benefit from them,” said Justin Taylor, M.D., a Sylvester physician-scientist and co-senior author of the study.

Related Links
University of Miami Miller School of Medicine