Single-Cell Nucleic Acid Normalization via Exogenous Reference
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods lack effective normalization approaches for nucleic acid and protein analysis at the single-cell level, particularly for tumor suppressor gene activity and oncogene copy number alterations, which is crucial for cancer research and diagnostics.
Innovation Solution
The use of microfluidic, droplet-based technology with PCR-based assays and sample normalization components, including exogenous nucleic acids with known sequences, to normalize nucleic acid concentrations and amplify target sequences, allowing for robust correction of PCR efficiency and characterization of single cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standardized assay protocols are used for nucleic acid analysis, then processing efficiency is improved, but measurement precision across different samples deteriorates due to inability to account for sample-specific variability
Solution Approach 1:
The patent introduces normalization components that change the reference parameter from a fixed standardized value to a sample-specific normalized value. By adding normalization components with known concentrations and comparing their amplification to target sequences, the system adjusts PCR efficiency parameters for each sample individually, resolving the contradiction between standardized processing and precise measurement.
Solution Approach 2:
The patent uses normalization components as intermediary substances that mediate between the standardized protocol and sample-specific requirements. These components serve as internal controls that account for variability in nucleic acid extraction and PCR efficiency, allowing precise measurement while maintaining standardized processing workflows.
2Device complexity
If nucleic acid concentration is not normalized, then analysis simplicity is maintained, but reliability of quantitative readouts across samples deteriorates
Solution Approach 1:
The normalization component serves multiple functions: it acts as an internal control for nucleic acid concentration, a reference for PCR efficiency correction, and a standard for quantitative readout. This multi-functionality maintains analysis simplicity while significantly improving reliability across samples.
Solution Approach 2:
The system incorporates feedback by measuring the amplification of normalization components and using this information to correct target sequence quantification. The normalization component provides a reference signal that feeds back into the calculation of relative expression levels, ensuring reliable quantitative readouts.
3Measurement precision
If exogenous normalization components are added to each sample, then measurement precision is improved, but device complexity increases
Solution Approach 1:
The patent merges the normalization function with the existing PCR assay by combining target sequence amplification and normalization component amplification into a single multiplex reaction. This integration improves measurement precision while minimizing the increase in device complexity through consolidation of functions.
Solution Approach 2:
The normalization component creates a synthetic copy with known sequence and concentration that replicates the behavior of endogenous nucleic acids during PCR. This copying approach allows precise measurement without requiring complex modifications to the original sample processing workflow.
4Productivity
If single-cell analysis is performed without normalization, then analysis throughput is maintained, but manufacturing precision of quantitative data deteriorates
Solution Approach 1:
The normalization component is added to each single-cell sample during the initial processing step, before amplification occurs. This preliminary action ensures that normalization is built into the workflow from the start, maintaining high throughput while ensuring manufacturing precision of quantitative data through individual sample normalization.
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
Enables high-throughput, quantitative analysis of single cells, correcting for variability and characterizing nucleic acid and protein content, aiding in cancer subtype identification and diagnostics.
Implementation Method 1
providing a protease to each encapsulated cell and incubating the encapsulated cell with the protease to produce a cell lysate
Implementation Method 2
performing a nucleic acid amplification reaction to form an amplification product from the nucleic acid of a single cell
Implementation Method 3
providing a sample normalization component to one or more encapsulated cell, where the normalization component comprises an exogenous nucleic acid having a known sequence
Data Source
AI summary
Provided herein are methods and systems for detection of nucleic acids for single cell samples. As part of the detection, a unique step of normalization of different single cell samples is included. One embodiment of the method includes i) selecting one or more target nucleic acid sequence of interest in an individual cell, where the target nucleic acid sequence is complementary to a nucleic acid in a cell; ii) providing a sample having a plurality of individual single cells and encapsulating one or more individual cell(s); iii) providing a sample normalization component to one or more encapsulated cell, where the normalization component comprises an exogenous nucleic acid having a known sequence; iv) providing nucleic acid primers for the target nucleic acid and the exogenous nucleic acid; v) providing a protease to each encapsulated cell and incubating the encapsulated cell with the protease in the drop to produce a cell lysate; vi) performing a nucleic acid amplification reaction to form an amplification product from the nucleic acid of a single cell, where the amplification product comprise amplicons of one or more target nucleic acid sequence and an amplicon for the exogenous nucleic acid; and vii) comparing the amplification products from the target amplicons and the exogenous nucleic acid amplicons and determining the copy number or sequence of the target nucleic acid in a single cell.