Small RNA Expression Correction Using Long Nucleic Acid Standards
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Solution Overview
Problem
Current methods for correcting expression levels of target small RNAs in samples are inadequate, especially when dealing with small amounts of RNA or body fluids, as they fail to accurately account for extraction efficiency and are unstable due to sample conditions and impurities.
Innovation Solution
A method involving the addition of a nucleic acid standard substance with a length of at least 200 bases to samples, followed by extraction and measurement of both the target small RNA and standard substance, allowing for accurate correction of expression levels using a representative value and correction factor.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional correction methods (global normalization, quantile method, lowess method, or 75 percentile method) are used for small RNA expression analysis, then comprehensive detection of multiple genes is required, but these methods cannot be used when only particular genes are to be detected
Solution Approach 1:
The patent introduces a standard substance as an intermediary element that mediates the correction process. This standard substance is added to samples before extraction and serves as a reference to calculate correction factors, enabling accurate correction even when detecting only particular genes. The standard substance acts as a bridge between the extraction process and the final measurement, allowing correction without requiring comprehensive gene detection.
2Ease of operation
If house-keeping RNAs are used for correction in small RNA detection, then correction can be performed, but the expression levels of house-keeping RNAs are not constant among samples leading to inaccurate correction
Solution Approach 1:
The patent uses a standard substance that is added in known amounts to create a reference copy system. Instead of relying on endogenous house-keeping RNAs with variable expression, the method introduces an exogenous standard substance that serves as a stable reference. This standard substance is processed through the same extraction steps as the target small RNAs, allowing accurate calculation of correction factors based on the ratio between expected and actual recovery of the standard substance.
3Quantity of substance
If small amounts of RNA or body fluids are analyzed, then sample availability is improved, but extraction efficiency variations and sample conditions cause instability in measurement values
Solution Approach 1:
The patent implements a feedback mechanism by measuring the actual amount of standard substance recovered after extraction and using this information to calculate correction factors. The correction factor is determined by comparing the expected amount of standard substance (added in known quantities) with the actual measured amount, and this correction factor is then applied to correct the target small RNA expression levels. This feedback loop compensates for extraction efficiency variations and sample condition differences.
4Device complexity
If no correction is applied to account for extraction efficiency, then the analysis process is simpler, but the expression level measurements are inaccurate due to extraction variations among samples
Solution Approach 1:
The patent applies preliminary action by adding the standard substance to samples before the extraction process begins. This allows the standard substance to undergo the same extraction steps as the target small RNAs, so that any variations in extraction efficiency are captured in the standard substance recovery. The correction factor calculated from the standard substance then compensates for these extraction variations in the target RNA measurements.
Data Source
Figure 1A~1C
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AI summary
Means that enables, for comparative analysis of the expression level(s) of a target small RNA(s) among a plurality of samples, accurate correction of the measured value(s) of the expression level(s) utilizing a standard substance(s) is disclosed. In the correction method for the expression level(s) of a small RNA(s) according to the present invention, a nucleic acid(s) with a nucleic acid length of not less than 200 bases is/are used as the standard substance(s). A predetermined amount(s) of the standard substance(s) is/are added to a predetermined amount of each sample, and nucleic acid is extracted from the sample, followed by measuring the amounts of each target small RNA and the standard substance(s) extracted. By using the measured value(s) of the amount(s) of the standard substance(s) extracted, the expression level of each target small RNA is corrected. The present invention enables more accurate correction of the expression level(s) of a small RNA(s) among samples than conventional methods.