Single-Cell cDNA Library via Carrier Hybridization
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Solution Overview
Problem
Conventional methods for gene expression analysis at the single-cell level face challenges in detecting and quantifying mRNA due to low mRNA amounts, leading to limited analyzable genes and decreased sensitivity, especially with residual reagents inhibiting PCR amplification.
Innovation Solution
A method involving cell lysis and DNase treatment in a single tube, followed by hybridization of mRNA with oligo(dT) fixed on a carrier for reverse transcription, allowing for the creation of a single-cell derived cDNA library that can be amplified and reused for quantitative analysis without residual reagent inhibition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional RNA extraction and purification methods are used, then RNA can be extracted from multiple cells, but the extremely small amount of RNA (about 10 pg) contained in a single-cell cannot be recovered
Solution Approach 1:
The invention segments the RNA extraction process by performing cell lysis, DNase treatment, and reverse transcription in separate steps, with reverse transcription performed on a carrier surface. This segmentation allows for efficient recovery of extremely small amounts of RNA from single cells while maintaining ease of operation through standardized protocols for each step.
Solution Approach 2:
The invention introduces a carrier (such as magnetic beads or microtiter plate surface) as an intermediary surface for performing reverse transcription. This carrier serves as a mediator that concentrates the extremely small amount of RNA from single cells, enabling efficient recovery and subsequent analysis while simplifying the overall extraction and purification process.
2Productivity
If PCR amplification is performed after reverse transcription on magnetic beads, then cDNA can be amplified, but the absolute total number and ratio of RNA molecules are varied by the amplification step, making quantitative analysis impossible
Solution Approach 1:
The invention performs reverse transcription on a carrier surface before any amplification steps, creating a stable cDNA library that preserves the absolute total number and ratio of original RNA molecules. This preliminary action on the carrier establishes a quantitative reference that can be used for accurate measurement even after subsequent amplification steps.
Solution Approach 2:
The invention creates a copy of the RNA population in the form of cDNA on the carrier surface through reverse transcription. This cDNA copy maintains the quantitative information of the original RNA molecules, allowing subsequent PCR amplification to generate sufficient material for analysis while preserving the ability to perform accurate quantitative analysis by referencing the original cDNA copy.
3Ease of operation
If a reagent kit for single-cell gene expression analysis is used, then cell lysis, DNase treatment, and reverse transcription can be carried out in a single tube, but residual reagent remains and inhibits PCR amplification reaction
Solution Approach 1:
The invention extracts the reverse transcription step from the single-tube mixture and performs it on a carrier surface. This separation removes the harmful residual reagents from the reaction mixture before PCR amplification, eliminating inhibition while maintaining ease of operation through a simplified two-step process (reverse transcription on carrier, then PCR in separate tube).
Solution Approach 2:
The carrier serves as an intermediary that separates the reverse transcription step from the PCR amplification step. By performing reverse transcription on the carrier surface and then removing the carrier before PCR, the invention eliminates residual reagent inhibition while maintaining operational simplicity through a clean separation of steps.
4Object-affected harmful factors
If cDNA derived from a single-cell is divided into portions for analysis, then residual reagent inhibition can be reduced, but detection sensitivity of real-time PCR analysis greatly decreases
Solution Approach 1:
The invention extracts the cDNA from the residual reagent environment by performing reverse transcription on a carrier surface and then removing the carrier before PCR amplification. This extraction eliminates residual reagent inhibition completely, allowing the use of the entire cDNA sample for analysis without division, thereby maintaining maximum detection sensitivity.
5Productivity
If conventional methods are used to analyze gene expression in tissues and multiple cells, then variances between cells are averaged, but detailed information of living organism having dynamical changes cannot be obtained
Solution Approach 1:
The invention segments the analysis at the single-cell level by performing reverse transcription on individual cells captured on carrier surfaces. This segmentation preserves the unique gene expression profile of each cell rather than averaging across populations, allowing detailed analysis of individual cell variations while maintaining productivity through high-throughput carrier-based processing.
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 the efficient purification and recovery of small amounts of cDNA, maintaining quantification performance and allowing for the analysis of a wider variety of genes without sensitivity loss, even with small cell samples.
Implementation Method 1
hybridization of mRNA with oligo(dT) fixed on a carrier for reverse transcription
Implementation Method 2
DNase treatment in a single tube
Data Source
AI summary
It is an object to provide a method of suitably analyzing the amount of gene expression of a single-cell.A method of detecting a nucleic acid comprisinga step of sampling a single-cell from a sample containing at least a single-cell,a cell lysis step of lysing cell membrane of the sampled single-cell and extracting nucleic acids from the cell,a DNase treatment step of degrading DNA of the extracted nucleic acids with DNase,a step of hybridizing mRNA of the total RNA contained in the single-cell with oligo (dT) fixed onto a carrier,a step of performing reverse transcription of the mRNA hybridized with the oligo (dT) to fix cDNA derived from the single-cell onto the carrier, thereby preparing a single-cell derived cDNA library fixed onto a carrier, anda step of amplifying cDNA fixed onto the carrier and simultaneously detecting an amplification amount of the cDNA.