RNA Sequencing from Minimal Sample Volume via cDNA Library Construction
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Solution Overview
Problem
Traditional RNA sequencing methods require large biological samples and are inefficient, often leading to RNA degradation and inaccurate downstream sequencing due to complex and time-consuming processes.
Innovation Solution
A method for detecting RNAs in small biological samples by constructing a cDNA library using less than 1 mL of sample input, involving steps such as breaking up lipid bilayers, removing DNAs, synthesizing short cDNA oligonucleotides, ligating adaptors, and amplifying to generate a cDNA library for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional RNA sequencing methods are used, then sufficient RNA quantity can be obtained, but large biological samples are required and the process is complex and time-consuming
Solution Approach 1:
The patent extracts and isolates RNA molecules from complex biological samples through a streamlined process that removes unnecessary components (proteins, lipids, DNA) while preserving the target RNA molecules, enabling sequencing from minimal sample amounts without requiring complex multi-step purification procedures
Solution Approach 2:
The patent performs preliminary RNA isolation and library construction steps before sequencing, including RNA fragmentation, adapter ligation, and amplification, to prepare the sample in advance and eliminate the need for complex downstream processing while maintaining sequencing accuracy
2Quantity of substance
If traditional RNA sequencing methods are used, then adequate RNA can be isolated, but the process is time-consuming and RNA degradation occurs
Solution Approach 1:
The patent implements a continuous RNA sequencing workflow where RNA molecules are directly converted to cDNA libraries without interruption for purification or intermediate steps, maintaining constant processing action to eliminate time loss and prevent RNA degradation while ensuring adequate RNA quantity is maintained throughout
Solution Approach 2:
The patent skips unnecessary intermediate purification steps and direct processing stages, rushing through the workflow from RNA isolation to library construction in a condensed manner that reduces processing time and prevents degradation while maintaining sufficient RNA quantity for sequencing
3Productivity
If traditional RNA sequencing methods are used, then sequencing can be performed, but RNA degradation and unwanted modification occur
Solution Approach 1:
The patent uses cDNA as an intermediary molecule that stabilizes the RNA sequence information, converting the fragile RNA into more stable DNA form during library construction, which prevents RNA degradation and unwanted modifications while maintaining sequencing accuracy and enabling reliable downstream analysis
4Device complexity
If small biological samples are used, then sample quantity limitations are addressed, but sufficient RNA may not be obtained
Solution Approach 1:
The patent changes the processing parameters by using highly efficient RNA isolation reagents and optimized lysis conditions that maximize RNA recovery from minimal sample volumes, along with sensitive detection methods that can quantify and sequence RNA molecules even when starting with nanogram-level amounts from small biological samples
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 the detection of at least 100 different genes, including protein-coding and non-coding RNAs, from various sample types with high accuracy, using minimal sample volume, and effectively addresses the challenges of RNA degradation and sample quantity limitations.
Implementation Method 1
synthesizing a plurality of first strand cDNAs by reverse transcribing the RNAs in the biological sample
Implementation Method 2
removing deoxyribonucleic acids (DNAs) in the biological sample
Implementation Method 3
ligating at least one adaptor to an end of each short, double-stranded cDNA oligonucleotide
Data Source
AI summary
This invention provides methods for detecting RNAs in a biological sample while using only a small amount of sample input, e.g., less than or equal to 1 mL. The methods described herein are able to detect a large percentage of protein-coding genes having the ENSEMBL gene annotation HG38.