Multiplexed detection of RNA using MERFISH and branched DNA amplification
| Autor: | Jeffrey R. Moffitt, Xiaowei Zhuang, Chenglong Xia, Hazen P. Babcock |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Brightness lcsh:Medicine In situ hybridization Cellular imaging Multiplexing Article Fluorescence imaging Transcriptome 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Single-cell analysis Cell Line Tumor Image Processing Computer-Assisted medicine BDNA test Humans RNA Neoplasm lcsh:Science In Situ Hybridization Fluorescence Osteosarcoma Multidisciplinary medicine.diagnostic_test Fluorescence in situ hybridization Gene Expression Profiling lcsh:R RNA Fluorescence High-Throughput Screening Assays Wide-field fluorescence microscopy 030104 developmental biology chemistry Biophysics lcsh:Q Single-Cell Analysis Nucleic Acid Amplification Techniques 030217 neurology & neurosurgery DNA |
| Zdroj: | Scientific Reports, Vol 9, Iss 1, Pp 1-13 (2019) Scientific Reports |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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