biostudies-literatureZiegenhain CEuropean Molecular Biology OrganizationGöran Gustafssons Stiftelse för Naturvetenskaplig och Medicinsk ForskningHuman Frontier Science ProgramVetenskapsrådetVallee FoundationKnut och Alice Wallenbergs Stiftelse560-566https://www.ebi.ac.uk/biostudies/studies/S-EPMC9119855biostudies-literatureUnknown19(5)Single-cell sequencing methods rely on molecule-counting strategies to account for amplification biases, yet no experimental strategy to evaluate counting performance exists. Here, we introduce molecular spikes-RNA spike-ins containing built-in unique molecular identifiers (UMIs) that we use to identify critical experimental and computational conditions for accurate RNA counting in single-cell RNA-sequencing (scRNA-seq). Using molecular spikes, we uncovered impaired RNA counting in methods that were not informative for cellular RNA abundances due to inflated UMI counts. We further leverage molecular spikes to improve estimates of total endogenous RNA amounts in cells, and introduce a strategy to correct experiments with impaired RNA counting. The molecular spikes and the accompanying R pacNature methodsMolecular spikes: a gold standard for single-cell RNA counting.PMC91198552017-010622017.0110ALTF 673-2017LT000155/2017-LZiegenhain CSandberg RHendriks GJHagemann-Jensen MfalseMolecular spikes: a gold standard for single-cell RNA counting.Single-cell sequencing methods rely on molecule-counting strategies to account for amplification biases, yet no experimental strategy to evaluate counting performance exists. Here, we introduce molecular spikes-RNA spike-ins containing built-in unique molecular identifiers (UMIs) that we use to identify critical experimental and computational conditions for accurate RNA counting in single-cell RNA-sequencing (scRNA-seq). Using molecular spikes, we uncovered impaired RNA counting in methods that were not informative for cellular RNA abundances due to inflated UMI counts. We further leverage molecular spikes to improve estimates of total endogenous RNA amounts in cells, and introduce a strategy to correct experiments with impaired RNA counting. The molecular spikes and the accompanying R pac2022-01-01T00:00:00Z2022 May2026-05-27T20:30:26.936Z2025-02-19T02:43:55.152ZS-EPMC91198553546896710.1038/s41592-022-01446-x