Multiplex Detection of RNA Expression in Drosophila Embryos

  title={Multiplex Detection of RNA Expression in Drosophila Embryos},
  author={David Kosman and Claudia Mieko Mizutani and Derek Lemons and W Gregory Cox and William McGinnis and Ethan Bier},
  pages={846 - 846}
We present a fluorescence-based, multiplex in situ hybridization method that permits the simultaneous detection of five differently labeled antisense RNA probes and up to seven different transcripts in a single Drosophila embryo. We also show that it should be possible to increase the number of detected transcripts substantially with nascent transcript multiplex fluorescent in situ hybridization. These multiplex methods fill a current technological gap between high-resolution in situ… Expand
Fluorescent in situ hybridization protocols in Drosophila embryos and tissues.
This chapter describes optimized fluorescent in situ hybridization protocols for Drosophila embryos and tissues utilizing tyramide signal amplification, either for single genes or in a high-throughput format, which greatly increases the sensitivity, consistency, economy, and throughput of the procedure. Expand
Multicolor fluorescence RNA in situ hybridization of Drosophila brain tissue.
This protocol describes RNA in situ hybridization of Drosophila brain tissue using a fluorophore-conjugated tyramide that is easily made in the laboratory for a fraction of the cost of the commercially produced product. Expand
A multiplex fluorescent in situ hybridization protocol for clonal analysis of Drosophila oogenesis.
This work provides a protocol for the detection of RNA in Drosophila mosaic follicular epithelia, where the mosaic analysis with a repressible cell marker (MARCM) technique is used for expression of transgenes. Expand
Whole mount RNA fluorescent in situ hybridization of Drosophila embryos.
An optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos is described. Expand
High resolution fluorescent in situ hybridization in Drosophila.
  • E. Lécuyer
  • Biology, Medicine
  • Methods in molecular biology
  • 2011
This chapter describes a high-resolution FISH protocol for the detection of RNA expression and localization dynamics in embryos and tissues of the fruit fly, Drosophila melanogaster. Expand
High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification
The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages and allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time. Expand
Programmable in situ amplification for multiplexed imaging of mRNA expression
A multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR), in which RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. Expand
L-DNA-tagged fluorescence in situ hybridization for highly sensitive imaging of RNAs in single cells.
We report an effective fluorescence in situ hybridization strategy, named l-DNA tagged FISH (LT-FISH), for highly sensitive RNA detection in fixed cultured cells. LT-FISH includes two-stepExpand
Multiplex fluorescent in situ hybridization in zebrafish embryos using tyramide signal amplification.
This protocol can be extended to perform multiplex studies by repetition of the TSA-based detection for each target sequentially with a different fluorescent dye label, and it is demonstrated that this approach can be combined with standard horseradish peroxidase (HRP)-mediated immunocytochemistry procedures in addition to FISH. Expand
Visualization of Individual Scr mRNAs during Drosophila Embryogenesis Yields Evidence for Transcriptional Bursting
This work describes the detection and counting of transcripts within single cells of fixed, whole-mount Drosophila embryos via a combination of FISH, immunohistochemistry, and image segmentation, and presents novel evidence to show that it can robustly detect single mRNA molecules. Expand


A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback
A non-radioactive in situ hybridization technique for the localization of RNA in whole mount Drosophila embryos and revealed translational control of the maternally derived hb mRNA, which was difficult to detect by conventional techniques. Expand
Single-Cell Gene Expression Profiling
By combining advances in computational fluorescence microscopy with multiplex probe design, this work devised technology in which the expression of many genes can be visualized simultaneously inside single cells with high spatial and temporal resolution. Expand
Systematic determination of patterns of gene expression during Drosophila embryogenesis
Analyzing gene-expression patterns by in situ hybridization to whole-mount embryos provides an extremely rich dataset that can be used to identify genes involved in developmental processes that have been missed by traditional genetic analysis. Expand
Whole-Genome Analysis of Dorsal-Ventral Patterning in the Drosophila Embryo
Whole-genome microarray assays identified as many as 40 new Dorsal target genes, which encode a broad spectrum of cell signaling proteins and transcription factors, which represent one of the most extensive gene networks known for any developmental process. Expand
Transcribed genes are localized according to chromosomal position within polarized Drosophila embryonic nuclei
Using a high-resolution in situ hybridization method, it is found that each of 15 transcribed genes was localized as predicted by their chromosomal position and by the known polarized organization of the chromosomes. Expand
Directing cell division during development.
After this transition to zygotic control, pulses of string transcription define the timing of highly patterned embryonic cell divisions and cyclin accumulation is not rate limiting. Expand
Dynamic control of positional information in the early Drosophila embryo
This analysis implies that the threshold-dependent interpretation of maternal morphogen concentration is not sufficient to determine shifting gap domain boundary positions, and suggests that establishing and interpreting positional information are not independent processes in the Drosophila blastoderm. Expand
Supporting Online Material
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Genome Biol
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