miRNA Normalization Using let-7d, let-7g, and let-7i Reference Genes

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Solution Overview

Problem

Current methods for quantifying microRNAs (miRNAs) in clinical samples face challenges due to the lack of stable reference genes, leading to inaccuracies and unreliable normalization, especially in small sample sizes and varying experimental conditions.

Innovation Solution

The use of let-7d, let-7g, and let-7i miRNAs, or their combinations, as internal reference genes for normalization, along with auxiliary genes like U6, RNU44, and miR-16, to stabilize the quantification process through high-throughput sequencing and algorithms like geNorm and NormFinder.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If traditional reference genes (5SrRNA, 18SrRNA, U6) are used for miRNA normalization, then the detection process can be performed, but the accuracy and reliability are reduced due to extraction efficiency differences and inability to represent miRNA composition

Engineering Contradiction:

ImprovemiRNA quantification accuracyVSAvoidnormalization reliability

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent applies homogeneity by selecting reference genes (let-7d, let-7g, let-7i) that are endogenous miRNAs with similar attributes to the study subjects. These reference genes share the same biological characteristics, extraction efficiency, and representation of miRNA composition, eliminating the heterogeneity problem of using non-miRNA genes for normalization.

Inventive Principle:

Principle #33Homogeneity

2Ease of manufacture

If exogenous controls (artificially synthesized non-human miRNAs) are used for normalization, then the detection can be standardized, but they cannot correct discrepancies in sample collection and are not ideal reference genes

Engineering Contradiction:

Improvenormalization standardizationVSAvoidsample collection discrepancy correction

Core Design Contradiction:

Ease of manufactureVSReliability

Solution Approach 1:

The patent applies self-service by using endogenous miRNAs (let-7d, let-7g, let-7i) that are naturally present in the samples being studied. These reference genes automatically adapt to the specific sample conditions, extraction efficiency, and biological variability, providing self-correcting normalization without requiring external controls that may not match the sample characteristics.

Inventive Principle:

Principle #25Self-service

3Productivity

If any reference gene is used for miRNA normalization, then the quantification can proceed, but in small size samples the amount of total RNA is smaller than the limits of precise quantification by spectrophotometric method

Engineering Contradiction:

Improvequantification throughputVSAvoidsmall sample quantification precision

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent applies parameter changes by switching from spectrophotometric quantification (which has detection limits for small samples) to qRT-PCR-based quantification using endogenous miRNA reference genes. This methodological parameter change enables precise measurement of miRNA levels even in small sample sizes where total RNA amounts are below spectrophotometric detection thresholds.

Inventive Principle:

Principle #35Parameter changes

Data Source

PatentUS9353413B2Internal reference genes for microRNAs normalization and uses thereof

Publication Date: 2016.05.31 MICROMEDMARK BIOTECH CO LTD

AI summary

Disclosed are standardized reference genes for microRNAs and the use thereof. The reference gene is microRNA let-7d, let-7g, let-7i or a combination thereof. The reference genes have extremely high stability and accuracy compared to the currently most commonly used reference genes in microRNA quantitation.