RNA Quantification via Spike-in Molecular Labels
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Solution Overview
Problem
Current methods for assessing RNA purity and quantity often fail to accurately measure the functional integrity of RNA samples, which are crucial for downstream applications, especially with precious and limited samples.
Innovation Solution
The method involves hybridizing oligonucleotides with target-specific regions to mRNA molecules, using spike-in RNA with molecular labels, and determining the functional integrity by counting the number of associated labels through reverse transcription and amplification, allowing for sensitive and accurate measurement of RNA integrity even in low quantities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If spectrophotometric analysis or UV fluorescence is used to estimate RNA quantity, then the measurement process is simple and quick, but the measurement precision and functional integrity assessment are insufficient
Solution Approach 1:
The patent introduces spike-in RNA molecules with molecular labels as intermediaries to bridge the gap between simple measurement and accurate functional integrity assessment. These labeled RNA molecules are added to the sample, undergo reverse transcription with the sample RNA, and their label counts serve as a reference to calculate reverse transcription efficiency, enabling precise functional integrity measurement without complex instrumentation
Solution Approach 2:
The patent creates digital copies of RNA molecule counts through molecular label sequencing. By counting the number of molecular labels associated with spike-in RNA and reference genes, the system generates digital representations of RNA quantity and quality, transforming physical RNA characteristics into countable digital data for precise functional integrity assessment
2Reliability
If gel electrophoresis or Bioanalyzer is used to check RNA fragmentation, then physical integrity can be assessed, but precious and limited RNA samples are consumed
Solution Approach 1:
The patent creates digital copies of RNA integrity information through molecular label counting. Instead of physically analyzing RNA fragments that would consume the sample, the method sequences and counts molecular labels from spike-in RNA that co-underwent reverse transcription, generating digital data about reverse transcription efficiency as a proxy for functional integrity without additional sample consumption
Solution Approach 2:
The patent makes the RNA sample self-serve the integrity assessment function. The same RNA sample used for downstream applications simultaneously serves as the substrate for integrity assessment, because the reverse transcription efficiency measured through spike-in RNA labels directly reflects the functional quality of the sample RNA without requiring separate assessment procedures
3Measurement precision
If molecular labels with high diversity are used to count RNA molecules, then the measurement precision and sensitivity are improved, but the device complexity and labeling cost increase
Solution Approach 1:
The patent replaces complex physical labeling systems with a simplified molecular label approach. Instead of using diverse fluorescent tags requiring complex detection equipment, the method uses nucleotide sequence-based molecular labels that are naturally diverse through stochastic incorporation during reverse transcription, and can be counted through standard sequencing or amplification techniques with simpler instrumentation
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 precise determination of RNA functional integrity, improving the assessment of RNA quality and efficiency of reverse transcription, even with small amounts of RNA, thereby optimizing downstream applications.
Implementation Method 1
hybridizing a plurality of oligonucleotides comprising a target specific region that specifically binds to a target in an mRNA molecule
Implementation Method 2
The extending step comprises reverse transcription of the mRNA molecule
Data Source
AI summary
Provided herein are methods, compositions, systems, devices, and kits for quantification of RNA, and determination of efficiency of reverse transcription of RNA to cDNA.