A novel RNA sequencing technology enables simultaneous quantification of intracellular proteins and transcriptomes, which could transform cancer research and therapeutics. Scientists at Sylvester Comprehensive Cancer Center (FL, USA), working alongside collaborators at the University of California, San Francisco (CA, USA) and the Helen Diller Family Comprehensive Cancer Center (CA, USA), have introduced CIPHER-seq (Cytokine Intracellular Protein High-throughput Expression with RNA-sequencing) – a new single-cell technology that can quantify RNA and intracellular proteins simultaneously, providing a more complete view of real-time immune signaling that could help further our understanding of disease and improve immunotherapy design. Single-cell RNA sequencing measures whole transcriptome gene expression in individual cells but fails to predict protein abundance accurately, particularly for cytokines. This means that direct protein measurement is required alongside transcriptome quantification. Despite recent advances in surface-protein CITE-seq, which have enabled extensive multimodal profiling, and developments in spatial technologies that allow tissue-based mapping, intracellular adaptation remains a challenge for suspension-based populations. On top of this, standard staining protocols optimized for flow cytometry often degrade RNA or elevate mitochondrial transcripts, compromising sequencing quality, while commercial intracellular protocols frequently introduce cellular stress artifacts. As a result, the team developed CIPHER‑seq, integrating optimized fixation chemistry, controlled permeabilization, abbreviated antibody incubation and RNase-protective conditions to measure intracellular proteins and transcriptomes concurrently with fewer disadvantageous consequences. Developments in digital PCR: finding the needle in a haystack Digital PCR (dPCR) is the third generation of PCR technology, after conventional PCR and real-time quantitative PCR. It offers significant benefits over these other methods,…
