Multiplexed detection of RNA using MERFISH and branched DNA amplification

• Published in: Scientific reports Vol. 9; no. 1; p. 7721
• Main Authors: Xia, Chenglong, Babcock, Hazen P., Moffitt, Jeffrey R., Zhuang, Xiaowei
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 22.05.2019; Nature Publishing Group
• Subjects: 14; 14/32; 14/63; 38/39; 631/1647/2017/1947; 631/1647/2217/2018; 631/1647/245/2225; 631/1647/328/2236; 631/80/2373; Binding sites; Brightness; Cell culture; Cell division; Cell Line, Tumor; Chemistry; Deoxyribonucleic acid; DNA; DNA probes; Error correction & detection; Fluorescence in situ hybridization; Gene expression; Gene Expression Profiling - methods; High-Throughput Screening Assays; Humanities and Social Sciences; Humans; Hybridization; Image Processing, Computer-Assisted; In Situ Hybridization, Fluorescence - methods; Indigenous species; multidisciplinary; Nucleic Acid Amplification Techniques - methods; Osteosarcoma - pathology; Ribonucleic acid; RNA; RNA - analysis; RNA, Neoplasm - analysis; Science; Science (multidisciplinary); Single-Cell Analysis
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-43943-8

Multiplexed error-robust fluorescence in situ hybridization (MERFISH) allows simultaneous imaging of numerous RNA species in their native cellular environment and hence spatially resolved single-cell transcriptomic measurements. However, the relatively modest brightness of signals from single RNA molecules can become limiting in a number of applications, such as increasing the imaging throughput, imaging shorter RNAs, and imaging samples with high degrees of background, such as some tissue samples. Here, we report a branched DNA (bDNA) amplification approach for MERFISH measurements. This approach produces a drastic signal increase in RNA FISH samples without increasing the fluorescent spot size for individual RNAs or increasing the variation in brightness from spot to spot, properties that are important for MERFISH imaging. Using this amplification approach in combination with MERFISH, we demonstrated RNA imaging and profiling with a near 100% detection efficiency. We further demonstrated that signal amplification improves MERFISH performance when fewer FISH probes are used for each RNA species, which should allow shorter RNAs to be imaged. We anticipate that the combination of bDNA amplification with MERFISH should facilitate many other applications and extend the range of biological questions that can be addressed by this technique in both cell culture and tissues.