Abstract 1940: High-throughput imaging and high content analysis of disease relevant lncRNAs examined by RNA fluorescence in situ hybridization (RNA FISH).

2013 ◽  
Author(s):  
Hans E. Johansson ◽  
Arturo V. Orjalo ◽  
Sally R. Coassin ◽  
Jerry. L. Ruth ◽  
Fabio Stossi ◽  
...  
Keyword(s):  
Content Analysis ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
High Throughput ◽  
Rna Fish ◽  
High Content Analysis ◽  
High Throughput Imaging

An Automated High-Throughput Fluorescence In Situ Hybridization (FISH) Assay Platform for Use in the Identification and Optimization of siRNA-Based Therapeutics

2020 ◽  
pp. 247255522096004
Author(s):  
Hui H. Dou ◽  
Rommel Mallari ◽  
Andrew Pipathsouk ◽  
Amrita Das ◽  
Mei-Chu Lo
Keyword(s):  
In Situ Hybridization ◽  
Gene Silencing ◽  
Fluorescence In Situ Hybridization ◽  
High Throughput ◽  
Messenger Rna ◽  
Great Promise ◽  
Small Interfering Rnas ◽  
Response Curves ◽  
Vitro Assay

Since the revolutionary discovery of RNA interference (RNAi) more than 20 years ago, synthetic small interfering RNAs (siRNAs) have held great promise as therapeutic agents for treating human diseases by the specific knockdown of disease-causing gene products. To facilitate the development of siRNA therapeutics, a robust, high-throughput in vitro assay for measuring gene silencing is imperative during the initial siRNA lead sequence identification and, later, during the lead optimization with chemically modified siRNAs. There are several potential assays for measuring gene expression. Quantitative reverse transcription PCR (qRT-PCR) has been widely used to quantitate messenger RNA (mRNA). This method has a few disadvantages, however, such as the requirement for RNA isolation, complementary DNA (cDNA) generation, and PCR reaction, which are labor-intensive, limit the assay throughput, and introduce variability. We chose a high-content imaging assay, bDNA FISH, that combines the branched DNA (bDNA) technology with fluorescence in situ hybridization (FISH) to measure gene silencing by siRNAs because it is sensitive and robust with a short reagent procurement and assay development time. We also built a fully automated liquid-handling platform for executing bDNA FISH assays to increase throughput, and the system has a capacity of generating 192 concentration–response curves in a single run. We have successfully developed and executed the bDNA FISH assays for multiple targets using this automated platform to identify and optimize siRNA candidate molecules. Examples of the bDNA FISH assay for selected targets are presented.

scholarly journals Cytoplasmic DNA can be detected by RNA fluorescence in situ hybridization

2019 ◽  
Vol 47 (18) ◽  
pp. e109-e109
Author(s):  
Eliraz Greenberg ◽  
Hodaya Hochberg-Laufer ◽  
Shalev Blanga ◽  
Noa Kinor ◽  
Yaron Shav-Tal
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Sequences ◽  
Plasmid Dna ◽  
Double Helix ◽  
Rna Molecules ◽  
Rna Fish ◽  
Dna Double Helix ◽  
Intracellular Detection

Abstract Fluorescence in situ hybridization (FISH) can be used for the intracellular detection of DNA or RNA molecules. The detection of DNA sequences by DNA FISH requires the denaturation of the DNA double helix to allow the hybridization of the fluorescent probe with DNA in a single stranded form. These hybridization conditions require high temperature and low pH that can damage RNA, and therefore RNA is not typically detectable by DNA FISH. In contrast, RNA FISH does not require a denaturation step since RNA is single stranded, and therefore DNA molecules are not detectable by RNA FISH. Hence, DNA FISH and RNA FISH are mutually exclusive. In this study, we show that plasmid DNA transiently transfected into cells is readily detectable in the cytoplasm by RNA FISH without need for denaturation, shortly after transfection and for several hours. The plasmids, however, are usually not detectable in the nucleus except when the plasmids are efficiently directed into the nucleus, which may imply a more open packaging state for DNA after transfection. This detection of plasmid DNA in the cytoplasm has implications for RNA FISH experiments and opens a window to study conditions when DNA is present in the cytoplasm.

scholarly journals Use of RNA Fluorescence In Situ Hybridization in the Prenatal Molecular Diagnosis of Myotonic Dystrophy Type I

2006 ◽  
Vol 52 (2) ◽  
pp. 319-322 ◽  
Author(s):  
Emanuela Bonifazi ◽  
Francesca Gullotta ◽  
Laura Vallo ◽  
Raniero Iraci ◽  
Anna Maria Nardone ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Myotonic Dystrophy ◽  
Somatic Mosaicism ◽  
Type I ◽  
Myotonic Dystrophy Type ◽  
Rna Fish ◽  
Ctg Expansion ◽  
Nuclear Foci

Abstract Background: Myotonic dystrophy type 1 (DM1; OMIM #160900) is an autosomal-dominant genetic disorder with multisystemic clinical features associated with a CTG expansion in the 3′ untranslated region of the DMPK gene on chromosome 19q13.3. A long-PCR protocol to detect the DM1 expansion is rapid, sensitive, and accurate, but interpretative limitations can occur when the expansion size exceeds the PCR amplification range and in cases of somatic mosaicism. Methods: To overcome these problems, we used RNA fluorescence in situ hybridization (RNA-FISH) to study cultured cells derived from chorionic villus samples (CVS) with the DM1 mutation. The RNA-FISH method is designed to detect the distinctive DM1 cellular phenotype, characterized by the presence of nuclei with focal ribonuclear inclusions (foci) containing the DMPK expanded transcripts. We analyzed 6 CVS from DM1-predicted pregnancies and 6 CVS from DM1-negative pregnancies. Results: In 4 DM1-predicted fetuses with a CTG expansion >200 CTG, varying numbers of ribonuclear inclusions were clearly visible in all cells. One case with a somatic mosaicism for the DMPK mutation showed 15% of cells with no nuclear foci. No nuclear signals were detected in all controls examined (n = 6) and in 1 DM1-positive sample with a CTG expansion <100 copies. Conclusion: Nuclear foci, and therefore the DM1 mutation they are caused by, can be detected efficiently on interphase nuclei of trophoblast cells with RNA-FISH when the CTG expansion is >200 copies.

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