Affinity-Tagged Antisense Probes for rRNA Depletion in FFPE Samples
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Solution Overview
Problem
Current methods are inadequate for effectively removing degraded rRNA from samples, particularly from formalin-fixed paraffin-embedded (FFPE) tissue sections, which hinders analysis of other RNA molecules of interest in gene expression studies and clinical diagnostics.
Innovation Solution
The development of affinity-tagged antisense rRNA molecules complementary to specific rRNA sequences, which are used to generate rRNA-depleted samples or isolate rRNA, utilizing a binding matrix to remove rRNA molecules, thereby enhancing the analysis of non-rRNA molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional methods are used to remove rRNA from samples, then some rRNA removal is achieved, but degraded rRNA from FFPE tissue sections cannot be effectively removed
Solution Approach 1:
The patent employs RNase H enzyme which recognizes RNA-DNA hybrid structures and selectively degrades the RNA component. This enzymatic approach changes the mechanism from physical/chemical removal to biological recognition, enabling effective degradation of rRNA including fragmented forms in FFPE samples while preserving other RNA molecules
Solution Approach 2:
The invention uses oligonucleotide probes as intermediaries that hybridize to target rRNA sequences. These probes serve as mediators between the detection system and the rRNA molecules, enabling specific recognition and subsequent removal of rRNA through RNase H-mediated degradation
2Stability of the object's composition
If rRNA is present in samples, then the natural RNA composition is maintained, but analysis of other RNA molecules is complicated
Solution Approach 1:
The patent selectively removes rRNA from the total RNA population using RNase H-mediated degradation. This extraction of the problematic component (rRNA) allows the remaining RNA molecules to be analyzed without interference, improving measurement precision for gene expression studies while maintaining the integrity of the original sample composition through targeted removal
3Ease of manufacture
If degraded rRNA from FFPE samples is not effectively removed, then sample preparation is simplified, but valuable clinical information remains inaccessible
Solution Approach 1:
The RNase H enzyme performs self-service by automatically recognizing and degrading rRNA sequences through its catalytic activity. The system uses the complementary base pairing between oligonucleotide probes and rRNA to guide the enzymatic degradation, eliminating the need for complex manual intervention while effectively removing degraded rRNA to recover clinical information
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 approach allows for the generation of rRNA-depleted samples that are substantially free of rRNA sequences, enabling more accurate analysis of non-rRNA molecules, such as mRNA, miRNA, and other transcriptomic components, improving diagnostic and therapeutic applications.
Implementation Method 1
affinity-tagged antisense rRNA molecules that exhibit sequences complementary to substantially all of at least one full-length rRNA molecule
Implementation Method 2
utilizing a binding matrix to remove rRNA molecules
Data Source
AI summary
The present invention provides methods, compositions, and kits for generating rRNA-depleted samples and for isolating rRNA from samples. In particular, the present invention provides compositions comprising affinity-tagged antisense rRNA molecules corresponding to substantially all of at least one rRNA molecule (e.g., 28S, 26S, 25S, 18S, 5.8S and 5S eukaryotic cytoplasmic rRNA molecules, 12S and 16S eukaryotic mitochondrial rRNA molecules, and 23S, 16S and 5S prokaryotic rRNA molecules) and methods for using such compositions to generate rRNA-depleted samples or to isolate rRNA molecules from samples.