Co-detecting mRNA and miRNA via Nuclease Protection Probes
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Solution Overview
Problem
Current methods struggle to simultaneously and accurately detect messenger RNA (mRNA) and small non-coding RNA, such as microRNA (miRNA), due to differences in probe binding kinetics and sample preparation challenges, which limits comprehensive transcriptome analysis.
Innovation Solution
The development of a quantitative nuclease protection assay (qNPA) that allows for the simultaneous detection of mRNA and miRNA in the same sample using nuclease protection probes (NPPs) of similar lengths, eliminating the need for extensive sample preparation and reducing cross-sample artifacts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If separate detection methods are used for mRNA and miRNA, then detection accuracy for each RNA type can be optimized, but sample-to-sample artifacts increase and comprehensive transcriptome analysis becomes difficult
Solution Approach 1:
The patent combines separate detection methods for mRNA and miRNA into a unified assay platform. By using a common probe design system with RNA polymerase I transcription-based probes that can detect both mRNA and miRNA through the same hybridization and detection workflow, the method eliminates sample-to-sample artifacts while maintaining detection accuracy for both RNA types simultaneously.
2Measurement precision
If different probe lengths are used for mRNA and miRNA detection, then each RNA type can be detected with optimal probe binding, but assay complexity increases and simultaneous detection becomes difficult
Solution Approach 1:
The patent changes the probe length parameter to a universal range of 15-30 nucleotides that works optimally for both mRNA and miRNA detection. This parameter standardization allows both RNA types to be detected with the same probe design rules and assay conditions, eliminating the need for separate optimized protocols while maintaining high detection precision for each RNA type.
3Measurement precision
If extensive sample preparation is performed to separate mRNA and miRNA, then detection specificity can be improved, but interassay variability increases and processing time increases
Solution Approach 1:
The patent extracts and removes the extensive sample preparation steps that were previously necessary to separate mRNA and miRNA for individual detection. By using a unified detection approach where both RNA types can be detected directly from the same sample extract without separation, the method maintains detection specificity while eliminating time-consuming preparation steps and reducing interassay variability.
4Adaptability or versatility
If multiple separate assays are used to detect multiple mRNAs and miRNAs, then comprehensive transcriptome analysis can be achieved, but interassay variability increases and throughput decreases
Solution Approach 1:
The patent creates a universal detection platform that can simultaneously detect multiple mRNA and miRNA species in a single assay. The standardized probe design and detection workflow allow the same assay system to be applied to any RNA target, enabling comprehensive transcriptome analysis with high throughput and eliminating interassay variability that plagues multiple separate assays.
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 enables highly sensitive and specific co-detection of multiple mRNAs and miRNAs in a single assay, reducing background noise and interassay variability, and allowing for single copy gene detection in minimal cell samples without the need for amplification.
Implementation Method 1
contacting a sample with a plurality of nuclease protection probes (NPPs) including at least one NPP which specifically binds to a target mRNA and at least one NPP which specifically binds to a target small non-coding RNA
Implementation Method 2
The sample is contacted with a nuclease specific for single-stranded nucleic acid molecules under conditions sufficient to remove unbound nucleic acid molecules
Data Source
AI summary
Disclosed herein are methods of co-detecting presence of target messenger RNA (mRNA) and small non-coding RNA (for example, miRNA) in a sample. The disclosed methods can be used to simultaneously detect mRNA and small non-coding RNA in a single assay (for example in the same reaction or the same well of a multi-well assay). The methods can include contacting a sample with a plurality of nuclease protection probes (NPPs) including at least one probe which specifically binds to a target mRNA and at least one probe which specifically binds to a target small non-coding RNA, contacting the sample with a nuclease specific for single-stranded nucleic acids, and detecting the NPP, for example on a microarray.