Modular Microfluidic Droplet System for Full-Length Total RNA Analysis
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Solution Overview
Problem
Current droplet microfluidic methods for RNA analysis from single cells only analyze messenger RNA (mRNA) and some long non-coding RNAs, failing to capture the full length of RNA, which is essential for understanding alternative splicing and RNA velocity determination.
Innovation Solution
A microfluidic method and system for preparing sequencing libraries that encapsulates cells or cell structures in droplets, releases and optionally fragments RNA, adds oligonucleotide tags, and performs reverse transcription to obtain cDNA libraries with barcodes and Unique Molecular Identifiers (UMIs), enabling the analysis of full-length total RNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current droplet microfluidic methods are used for RNA analysis, then the analysis process is simplified and throughput is improved, but only mRNA and some long non-coding RNAs can be analyzed and full length RNA information is lost
Solution Approach 1:
The method segments the RNA analysis process into distinct microfluidic droplet stages: encapsulation of single cells, lysis to release RNA, optional fragmentation into manageable pieces, tagging with oligonucleotide adapters, and reverse transcription to cDNA. This segmentation allows full-length RNA analysis to be achieved through systematic processing of fragmented RNA pieces, each retaining barcode information for reconstruction of the complete transcript sequence and length information.
2Device complexity
If current droplet microfluidic methods analyze only mRNA and some long non-coding RNAs, then the method complexity is reduced, but the versatility of RNA type analysis is limited
Solution Approach 1:
The microfluidic method achieves universality by using a common workflow applicable to all RNA types. The lysis step releases all RNA types from cells, the optional fragmentation step handles different RNA sizes uniformly, and the tagging step adds universal oligonucleotide adapters that enable subsequent reverse transcription and sequencing for any RNA molecule, whether mRNA, non-coding RNA, or other RNA types.
3Measurement precision
If full length RNA analysis is implemented, then insights into alternative splicing and RNA velocity are improved, but the processing complexity and reagent requirements increase
Solution Approach 1:
The method applies preliminary action by adding oligonucleotide tags to the RNA molecules during the early stages of processing, before fragmentation and reverse transcription. These tags are incorporated into the final cDNA library and enable precise measurement of RNA length and full-length reconstruction without requiring complex post-processing steps. The barcode sequences are established early and carried through all subsequent operations.
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 allows for the comprehensive analysis of various RNA types, including those not previously accessible, providing detailed insights into gene expression and alternative splicing, and improving the accuracy of RNA velocity measurements.
Implementation Method 1
encapsulating in a microfluidic droplet: a cell or cell structure comprising RNA; and lysis and optionally RNA fragmentation reagent
Implementation Method 2
adding an RNA tagging reagent into the droplet, wherein the RNA tagging reagent adds an oligonucleotide tag to the RNA
Implementation Method 3
hybridizing the oligonucleotide tag of the RNA to a primer adapted to initiate cDNA synthesis
Implementation Method 4
performing reverse transcription to obtain a cDNA sequencing library wherein the cDNA in the cDNA sequencing library comprise a barcode and optionally a UMI
Data Source
AI summary
Modular microfluidic devices, systems and methods for total RNA analyses The invention relates to microfluidic methods of preparing a sequencing library for analyses of total RNA. The invention also relates to modular microfluidic systems for carrying out these methods.