Beyond the RNA Census: A Literature-Derived Measurement-Claim Framework for Low-Copy, Transient, Spatial, and Native-State RNA Analysis

An analytical envelope, not biotype, sets a class of RNA measurement problem: one to a hundred copies per cell, lifetimes of minutes to one or two hours, or confinement finer than a dissociated cell. Inside it, short-read endpoint sequencing forfeits a figure of merit at a specific step, and information discarded upstream cannot be reconstructed downstream. This review audits that workflow stage by stage, then assesses nine modality families. The evidence base was assembled by a structured but non-exhaustive search: 40 query axes in PubMed, extended by targeted searches of every unfilled cell, 43 axes in OpenAlex, and a wider date window. Retrieval was capped, one author screened, and every supporting record appraised on five items. The outcome is a literature-derived decision framework that infers, rather than demonstrates, its ordering: a measurement claim returns the quantity to preserve, the modalities that preserve it, the disqualifying failure mode, and the recommended validation. Among the modalities represented in the retrieved evidence, none was shown to maximize counting fidelity, spatial coverage, absence of measurement-induced perturbation of a live process, and native-state preservation together. Among the literature retrieved here, no commutable reference material establishes traceability between in situ counting and extract-based quantification.