In Situ Cloning From Fixed Tissue Using Oligonucleotide Probes
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Solution Overview
Problem
Current methods for analyzing RNA from fixed tissue samples are hindered by short RNA sequences, making isolation and analysis difficult, and existing techniques for gene expression studies in fixed tissues are less accurate due to dilution by non-malignant cells and loss of tissue architecture.
Innovation Solution
A method involving oligonucleotide sequence probes with degenerate sequence tags for in situ hybridization and cloning of nucleic acids from fixed biological samples, allowing for the isolation and amplification of RNA without the need for RNA extraction or in situ PCR, enabling gene expression analysis in fixed tissues.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If cross-linking fixative is used to preserve cellular morphology and constituents, then tissue morphology is retained, but RNA sequences become short and difficult to isolate and analyze
Solution Approach 1:
The invention changes the chemical parameters of the fixative by using paraformaldehyde instead of traditional cross-linking fixatives like glutaraldehyde. Paraformaldehyde provides morphological preservation while minimizing RNA cross-linking, thereby maintaining RNA integrity and enabling successful isolation and analysis.
Solution Approach 2:
The invention converts the potential harm of fixative-induced RNA degradation into a benefit by selecting a fixative (paraformaldehyde) that preserves morphology without severely compromising RNA quality. The fixed tissue can still yield sufficient RNA for gene expression analysis, turning a previously unusable sample type into a valuable resource.
2Ease of manufacture
If disaggregation of the sample is performed for Southern, Northern, or Western blot analysis, then nucleic acid extraction is enabled, but accuracy is reduced due to dilution of malignant cells by non-malignant cells
Solution Approach 1:
The invention extracts only the necessary information (RNA from malignant cells) without requiring complete disaggregation of the tissue. By using in situ hybridization and laser capture microdissection, RNA is isolated directly from malignant cells within the tissue architecture, eliminating dilution by non-malignant cells while maintaining extraction feasibility.
Solution Approach 2:
The invention applies local quality by treating different regions of the tissue differently. Malignant cells are selectively identified and processed for RNA isolation, while surrounding non-malignant cells are excluded. This localized approach ensures high measurement precision by focusing analysis on the relevant cell population.
3Ease of manufacture
If disaggregation of the sample is performed, then nucleic acid analysis is enabled, but tissue architecture is lost and correlation of genetic abnormalities with malignant cells becomes impossible
Solution Approach 1:
The invention performs preliminary action by preserving tissue architecture through careful fixation and processing before RNA isolation. The tissue is maintained in its structural context, allowing subsequent correlation of genetic abnormalities with specific morphological features and cell locations.
Solution Approach 2:
The invention uses a nested approach where RNA isolation and analysis are performed within the context of preserved tissue architecture. Laser capture microdissection allows selective extraction of RNA from specific cells while maintaining the spatial relationship between cells and tissue structures, effectively nesting the molecular analysis within the histological context.
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
This method allows for robust RNA amplification and accurate gene expression profiling in fixed tissues, overcoming the limitations of short RNA sequences and cell dilution, and enabling the analysis of archival samples for disease research.
Implementation Method 1
hybridizing the population of oligonucleotide sequence probes with the nucleic acid in the biological specimen, thereby forming a population of hybridized oligonucleotide sequences probes
Data Source
AI summary
The present invention pertains to methods related to cloning nucleic acids from biological samples, particularly pathological tissue samples. This method includes hybridizing a population of oligonucleotide sequence probes comprising degenerate sequence tags to a fixed tissue, isolating the hybridized oligonucleotide sequence probes and amplifying the sequence tags in the hybridized oligonucleotide sequence probes. This method can be utilized to identify genes associated with disease and to quantitate the expression of disease-related transcripts. The method can also be used to identify truncated mRNAs.