Splinted Ligation Amplification for In Situ RNA Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Fixation methods like formaldehyde cross-linking complicate the isolation and analysis of nucleic acids from biological samples, making it difficult to analyze or localize DNA and RNA in situ within cells and tissues, which is crucial for molecular-level investigations.
Innovation Solution
A method involving hybridization of polynucleotide pairs complementary to non-overlapping sequences of target RNA in fixed biological samples, followed by ligation and amplification, allows for in situ analysis and localization of target RNA without releasing nucleic acids from the sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If fixation with cross-linking fixatives such as formaldehyde is used, then long-term preservation and archiving of samples is achieved, but isolation and analysis of nucleic acids becomes difficult
Solution Approach 1:
The patent introduces a bridge oligonucleotide as an intermediary molecule that binds to both the formalin-fixed RNA (through hybridization to complementary sequences) and the target RNA sequence of interest. This bridge oligonucleotide acts as a mediator that allows access to and amplification of target sequences within the cross-linked formalin-fixed tissue without requiring complete release of nucleic acids from the fixed matrix, thus resolving the contradiction between preservation and analyzability
2Reliability
If fixation is performed to preserve tissue structure, then histological assessment is enabled, but molecular-level analysis of nucleic acids is compromised
Solution Approach 1:
The patent segments the approach to nucleic acid analysis by using a bridge oligonucleotide that specifically targets and binds to particular RNA sequences within the fixed tissue. This segmentation allows molecular-level analysis of specific target sequences without requiring complete extraction or disruption of the fixed tissue structure, enabling both histological assessment and molecular analysis to proceed simultaneously
Solution Approach 2:
The bridge oligonucleotide serves as an intermediary that bridges the gap between the fixed tissue structure and the target RNA sequences. It hybridizes to complementary sequences in the formalin-fixed RNA and provides a handle for amplification and detection, allowing molecular analysis while preserving the overall tissue architecture for histological assessment
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables the amplification and detection of target RNA in situ, facilitating molecular analysis and localization within fixed biological samples, overcoming the limitations of traditional fixation methods.
Implementation Method 1
incubating the fixed biological sample with a pair of polynucleotides complementary to non-overlapping and proximal sequences of a target RNA, wherein the polynucleotide pair hybridizes to the target RNA
Implementation Method 2
ligating the polynucleotide pair using a ligase
Data Source
AI summary
The present invention relates to a method for amplifying at least one target RNA in a fixed and, optionally, expanded biological sample. In an embodiment of the invention, the method comprises incubating the fixed biological sample with a pair of polynucleotides complementary to non-overlapping and proximal sequences of a target RNA, wherein the polynucleotide pair hybridizes to the target RNA; ligating the polynucleotide pair using a ligase; and amplifying the ligation product. The invention further provides methods for detecting and optionally quantifying and/or sequencing the amplification product. As the method comprises hybridizing polynucleotide pairs to a target RNA in a fixed biological sample, the target RNA can be hybridized in situ.