Home › Biochemistry › Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay
Biochemistry JoVE (Open Access) Citable · DOI

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

DOI: 10.3791/58097-v
What you'll learn
  • ✓ Perform RNA in situ hybridization to detect sequences as short as 50 nucleotides
  • ✓ Achieve single-nucleotide resolution detection of genetic variants and point mutations
  • ✓ Visualize splice variants and short sequences within intact tissue morphology
  • ✓ Execute both manual and automated hybridization assay workflows
Protocol

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

Difficulty
advanced
Total time
~2–3 days (including tissue preparation, deparaffinization, hybridization, and detection)

Steps

1
Prepare tissue samples for in situ analysis

Obtain and prepare tissue specimens for hybridization assay. Ensure proper handling and storage to maintain tissue morphology and RNA integrity.

▶ 00:41
2
Assemble equipment and prepare reagents

Gather all necessary equipment and reagents required for the assay, including hybridization probes, detection reagents, and any automated equipment if applicable.

▶ 01:30
3
Deparaffinize, rehydrate, and pretreat tissue sections

Remove paraffin from tissue sections, rehydrate samples, and perform enzymatic or chemical pretreatment to expose RNA targets for probe binding.

▶ 03:24
4
Perform probe hybridization and signal amplification

Incubate tissue sections with complementary RNA probes and apply amplification chemistry to enhance detection sensitivity for short sequences and point mutations.

▶ 05:26
5
Counterstain, mount slide, and image tissue

Apply counterstains to visualize tissue morphology, mount slides with coverslips, and acquire high-resolution images for single-cell mutation and variant analysis.

▶ 07:18
6
Analyze detection of genetic variants and mutations

Examine results for successful visualization of splice variants, short sequences, and point mutations with single-nucleotide resolution in tissue context.

▶ 08:18
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