Cell-surface RNAs, including glycoRNAs, have been described, yet their precise composition across primary cell types remains unresolved, in part due to technical challenges in preserving membrane integrity and achieving specific detection. Here, we introduce a unified toolkit for profiling, imaging, and quantifying surface RNAs on intact cells. First, we present AMOUR, a T7-based linear amplification method that enables accurate, membrane-preserving profiling of surface RNAs. We then develop Intact-Surface-FISH, which labels target surface RNAs on live primary cells using fluorescent DNA probes. Coupled with super-resolution microscopy and flow cytometry, Intact-Surface-FISH allows robust visualization and quantification of representative surface RNAs on live primary cells.

Applying AMOUR, we chart the landscape of RNAs exposed on the outer plasma-membrane surface across diverse cell types and uncover a rich repertoire of noncoding RNAs on human and murine blood cells. Using Intact-Surface-FISH, we confirm membrane anchorage and quantify the abundance of multiple surface RNAs in cultured HeLa cells and human umbilical cord blood mononuclear cells (hUCB-MNCs). Imaging and flow cytometry jointly validate the membrane localization of Y-family RNAs, the spliceosomal snRNA U5, mitochondrial rRNA MTRNR2, mitochondrial tRNA MT-TA, VTRNA1-1, and the long noncoding RNA XIST. Three-dimensional, nanometer-scale imaging further corroborates the surface localization of RNY5, MTRNR2, and XIST on live hUCB-MNCs. Most binding proteins of the identified surface RNAs are reported autoantigens in autoimmune diseases. Most RNA-binding proteins associated with the identified surface RNAs have been reported as autoantigens in autoimmune diseases. Elucidating the biological functions of these unanticipated surface RNAs—and their potential contributions to autoimmunity—merits further investigation.

Together, these methodologies provide a rigorous framework for interrogating surface RNAs while maintaining plasma-membrane integrity and deliver a detailed reference of surface RNA landscapes in hUCB-MNCs and murine blood cells. This work establishes foundational tools and datasets that will accelerate mechanistic studies of surface RNAs, which are likely to assume diverse roles across cell types such as neurons, germ cells, and cancer cells.

The study present a suite of methods to accurately profile, image, and quantify cell-surface RNAs while preserving plasma-membrane integrity. High-resolution imaging and flow cytometry conclusively confirm the membrane localization of representative surface RNAs.
DOI：https://doi.org/10.1093/procel/pwaf079