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Fluorescent and Chromogenic In-Situ Hybridization Probes for Detecting Genetic Aberrations

Lab Protocols - Dartmouth College

1) . Test higher concentrations (e.g., start with one order of magnitude higher) to see if the signal quality improves. Based on experience, we recommend diluting in TE 1:20 for Cy5 coupled probes, 1:50 for Alexa, and 1:10 for TMR, followed by 1:100 dilution in hybridization buffer.

In Situ Hybridization of Whole-Mount Mouse Embryos with RNA Probes: Hybridization, Washes, ..

08/01/2018 · Procedure

A combination of scanning and imaging surface plasmon resonance (SPR) experiments is used to characterize DNA hybridization adsorption at gold surfaces and the subsequent immobilization of streptavidin. Single-stranded oligonucleotides are immobilized at gold surfaces, and the hybridization of biotinylated complements from solution is monitored with SPR. The subsequent attachment of streptavidin to the biotinylated complements provides a method of enhancing the SPR imaging signal produced as a result of the hybridization and leads to a 4-fold improvement in the hybridization detection limit of the SPR imaging apparatus. In situ scanning SPR experiments are used to measure a 60 ± 20% hybridization efficiency between immobilized single-stranded DNA and biotinylated complements. From the information provided by both the in situ imaging and scanning SPR experiments, an absolute surface coverage of immobilized single-stranded DNA is estimated to be ∼3 × 1012 molecules/cm2. The SPR signal resulting from hybridization onto immobilized probes is further amplified by the formation of streptavidin/DNA multilayers which grow by a combination of DNA hybridization and biotin−streptavidin binding. DNA/DNA multilayers without streptavidin are used as an additional method of amplifying the SPR signal.

3) . Formamide concentration of the hybridization buffer directly controls binding efficiency. While 10% formamide generally works well for all stages, one may try decreasing the concentration to see if signal improves. One should be cautious with this approach as low formamide concentration also ups the chance of non-specific binding.

Sequencing, forensic analysis and genetic analysis - …

In , the expression pattern of a gene provides important clues to understanding its biological function. To accurately depict endogenous transcriptional activity, a highly sensitive method is required to measure transcript levels in the intact tissue across various developmental stages. Conventional RNA hybridization methods using hapten- (biotin or digoxygenin) labeled RNA probes rely on antibody binding for visualization, and are thus only semi-quantitative at best (; ). Additionally, hapten-labeled probes are prone to diffuse localization (when conjugated with alkaline phosphatase), low sensitivity (when conjugated with fluorescent molecules), and non-specific probe binding. Here, we introduce a recently developed mRNA hybridization method () that circumvents the above difficulties to give single molecule resolution of transcript detection.

Surface Plasmon Resonance Imaging Measurements of …

Nonisotopically labeled probes have become increasingly popular in recent years and replace radioactive probes in techniques such as in situ hybridization, Southern, Northern and Western blot analysis. The advantages of these techniques are:

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One of the advantages of the DIG-System is the stability of the labeled probe. After hybridization against the blotted target, the hybridization solution still contains large amounts of unannealed DIG-labeled probe. Simply pour the solution into a plastic tube and store at –20° C for DNA probes and –70°C for RNA probes. DIG labeled probes are stable for at least 1 year when stored in this manner. For reuse, thaw and denature by heating to +95°C for 10 min. If the hybridization solution contains formamide or .

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The following protocol covers the 5 major steps of smFISH: Probe design and synthesis, Fixation of worms and embryos, Hybridization, Image acquisition, Data analysis. This protocol is largely adapted from the general smFISH protocol detailed in ), with notes and modifications specific to . Unless otherwise noted, all reagents listed can be made in bulk ahead of time and stored at room temperature (RT).