RNA Templated Ligation for Spatial Analysis Quality Assessment
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Solution Overview
Problem
Current methods for spatially analyzing RNA in biological samples, particularly FFPE tissues, face challenges due to RNA degradation and the need for sensitive detection of specific genes, often requiring resource-intensive sequencing and lacking information on the spatial location and abundance of target nucleic acids.
Innovation Solution
Targeted RNA capture methods are developed, which include using capture probes with spatial barcodes and capture domains to hybridize and ligate specific RNA molecules, allowing for sensitive measurement of specific genes and determination of their spatial location, optimized by assessing sample quality and permeabilization conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If poly(A) mRNA capture is used for spatial analysis, then a high number of analytes can be detected, but RNA degradation occurs and off-target nucleic acids are detected
Solution Approach 1:
The patent extracts the poly(A) tail from the mRNA molecule and uses it as a separate capture handle. The capture probe specifically binds to the poly(A) tail region, allowing selective enrichment of mRNA molecules while excluding other RNA types. This extraction approach enables specific target capture without detecting off-target nucleic acids.
Solution Approach 2:
The patent introduces an intermediary ligation step where a capture probe with poly(A) binding domain is ligated to the target mRNA. This intermediary mechanism ensures specific binding only to true poly(A) mRNA targets, filtering out off-target sequences while maintaining the ability to detect multiple analytes simultaneously.
2Measurement precision
If targeted RNA capture is used instead of poly(A) capture, then sensitivity for specific genes improves and RNA degradation is reduced, but the method complexity increases
Solution Approach 1:
The patent designs a universal capture probe system where a single probe structure with poly(A) binding domain can target any poly(A) mRNA in the transcriptome. This multi-functional approach allows the same basic probe design to detect thousands of different genes simultaneously, reducing method complexity while maintaining high sensitivity for specific genes.
Solution Approach 2:
The patent performs preliminary enrichment of poly(A) mRNA using capture probes before spatial analysis. This preliminary action concentrates the target RNA molecules and removes degradation products early in the workflow, improving sensitivity for specific gene detection while simplifying subsequent analysis steps.
3Loss of information
If sequencing is performed to determine spatial location and abundance, then comprehensive data is obtained, but time and resources are consumed
Solution Approach 1:
The patent replaces the mechanical sequencing process with a simplified detection approach. After targeted RNA capture and ligation, the spatial location and abundance information is obtained through direct detection methods rather than full sequencing, significantly reducing time and resource consumption while maintaining data completeness.
Solution Approach 2:
The patent extracts only the essential spatial and abundance information from the captured RNA molecules without performing complete sequencing. By taking out only the necessary data elements (spatial coordinates and signal intensity), the method achieves comprehensive spatial analysis with minimal time and resource investment.
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 enables efficient and sensitive detection of target RNA molecules with improved spatial resolution, reducing RNA degradation issues and enhancing the ability to detect specific genes of interest in FFPE tissues, while optimizing sample preparation for better data quality.
Implementation Method 1
using capture probes with spatial barcodes and capture domains to hybridize and ligate specific RNA molecules
Implementation Method 2
RNA-templated ligation offer an alternative to indiscriminant capture of a common transcript sequence
Data Source
AI summary
Provided herein are methods of testing a biological sample for efficacy of detection of a target nucleic acid where the method includes RNA templated ligation including generating a sequence that is complementary to the hybridized ligation product that includes labelled nucleotides and detecting a signal corresponding to the ligation product on the substrate, thereby determining the efficacy of detection of the target nucleic acid.