Year: 2025
Type: article
Abstract: Spatial transcriptomics has emerged as a powerful tool to define the cellular structure of diverse tissues. One such method is multiplexed error robust fluorescence in situ hybridization (MERFISH). MERFISH identifies RNAs with error tolerant optical barcodes generated through sequential rounds of single-molecule fluorescence in situ hybridization (smFISH). MERFISH performance depends on a variety of protocol choices, yet their effect on performance has yet to be systematically examined. Here we explore a variety of properties to identify optimal choices for probe design, hybridization, buffer storage, and buffer composition. In each case, we introduce protocol modifications that can improve performance, and we show that, collectively, these modified protocols can improve MERFISH quality in both cell culture and tissue samples. As RNA FISH-based methods are used in many different contexts, we anticipate that the optimization experiments we present here may provide empirical design guidance for a broad range of methods.
Source: Scientific Reports
Authors: Josh J. Luce, Christian A. Reardon-Lochbaum, Paolo Cadinu, Rosalind J. Xu, Jose Aceves-Salvador, Evangelia Semizoglou, Bertrand Wong, Iris Lopez, Alan B. Cantor, William Renthal, Jeffrey R. Moffitt
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,690
Awards: Bone Marrow Spatial Transcriptomics to Enhance In Vitro Platelet Production, Rapid, Robust, and Routine: Multiplexed Microscopy for Spatially Resolved Whole-Transcriptomic Single-Cell Profiling and the Construction of Cell Atlases of all Tissues and in all Organisms, Harvard PRECISION Human Pain Center, Pathophysiology of Human Blood Cells
OpenAlex ID: W4413862790