Luce Josh J, Reardon-Lochbaum Christian A, Cadinu Paolo, Xu Rosalind J, Aceves-Salvador Jose, Semizoglou Evangelia, Wong Bertrand, Lopez Iris, Cantor Alan B, Renthal William, Moffitt Jeffrey R
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, 02115, USA.
Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana- Farber Cancer Institute, Boston, MA, 02115, USA.
Sci Rep. 2025 Aug 31;15(1):32030. doi: 10.1038/s41598-025-17477-1.
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.
空间转录组学已成为定义多种组织细胞结构的强大工具。其中一种方法是多重误差稳健荧光原位杂交（MERFISH）。MERFISH通过连续多轮单分子荧光原位杂交（smFISH）生成的容错光学条形码来识别RNA。MERFISH的性能取决于多种实验方案的选择，但其对性能的影响尚未得到系统研究。在这里，我们探索了多种特性，以确定探针设计、杂交、缓冲液储存和缓冲液组成的最佳选择。在每种情况下，我们都引入了可以提高性能的实验方案修改，并且我们表明，总体而言，这些修改后的实验方案可以提高细胞培养和组织样本中MERFISH的质量。由于基于RNA FISH的方法在许多不同的背景中都有应用，我们预计我们在此展示的优化实验可能为广泛的方法提供经验性设计指导。
