期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：50
页码：14456-14461
DOI：10.1073/pnas.1617699113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceMultiplexed single-molecule FISH allows spatially resolved gene-expression profiling in single cells. However, because of off-target binding of FISH probes and cellular autofluorescence, background can become limiting in multiplexed single-molecule FISH measurements, especially when tissue samples are imaged or when the degree of multiplexing is increased. Here we report a sample clearing approach for FISH that substantially reduced these background sources by anchoring RNAs to a polymer matrix and then removing proteins and lipids. This approach allows measurements with higher detection efficiency and sensitivity across more color channels in both cell culture and tissue with no detectable loss in RNA. We anticipate that this clearing approach will greatly facilitate applications of multiplexed FISH measurements in a wide variety of biological systems. Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, background--from off-target binding of FISH probes and cellular autofluorescence--can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements. We identified off-target binding of FISH probes to cellular components other than RNA, such as proteins, as a major source of background. To remove this source of background, we embedded samples in polyacrylamide, anchored RNAs to this polyacrylamide matrix, and cleared cellular proteins and lipids, which are also sources of autofluorescence. To demonstrate the efficacy of this approach, we measured the copy number of 130 RNA species in cleared samples using multiplexed error-robust FISH (MERFISH). We observed a reduction both in the background because of off-target probe binding and in the cellular autofluorescence without detectable loss in RNA. This process led to an improved detection efficiency and detection limit of MERFISH, and an increased measurement throughput via extension of MERFISH into four color channels. We further demonstrated MERFISH measurements of complex tissue samples from the mouse brain using this matrix-imprinting and -clearing approach. We envision that this method will improve the performance of a wide range of in situ hybridization-based techniques in both cell culture and tissues.
