In Situ cDNA Library Construction Without RNA Isolation
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Solution Overview
Problem
Current RNA sequencing methods are limited by laborious RNA isolation steps, low throughput, and high costs, which hinder high-throughput gene expression analysis and drug screening.
Innovation Solution
A method called RNA Isolation Free Sequencing (RIF-Seq) that generates cDNA sequencing libraries in situ by immobilizing cells on a solid surface, performing reverse transcription within the cells using biotinylated dNTPs, releasing single-stranded cDNA without lysing the cells, and adding adapter molecules to facilitate library construction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional RNA isolation procedures are used, then RNA can be extracted and sequenced, but the process is laborious, low-throughput, and costly
Solution Approach 1:
The patent extracts only the essential function of RNA sequencing by performing reverse transcription directly in intact cells, eliminating the need to extract and isolate RNA. This removes the cumbersome RNA isolation step while preserving the core capability of converting RNA to cDNA for sequencing analysis.
Solution Approach 2:
The method enables multiple functions to be performed simultaneously: cells serve as both the biological sample and the reaction vessel, and the intact cell system supports both RNA preservation and cDNA synthesis. This multi-functionality increases throughput by eliminating separate isolation steps.
2Loss of time
If RNA isolation steps are performed, then RNA can be obtained for sequencing, but time is lost and cost increases
Solution Approach 1:
The patent performs reverse transcription action within intact cells before any isolation steps would be needed. By preparing the cDNA in situ within the cell, the method eliminates subsequent time-consuming isolation and purification steps, achieving both time and cost efficiency.
Solution Approach 2:
The intact cell serves as its own reaction vessel and protection mechanism. The cell's natural structure preserves RNA while the introduced reverse transcription machinery performs cDNA synthesis within the cell, eliminating the need for external isolation infrastructure and reducing costs.
3Adaptability or versatility
If cells are lysed to access RNA, then RNA can be extracted, but cell integrity is lost and further analysis is limited
Solution Approach 1:
The patent uses an intermediary approach by introducing reverse transcription primers and enzymes into intact cells that can access and convert RNA to cDNA without requiring cell lysis. This intermediary enzymatic system bridges the gap between intact cell preservation and RNA accessibility.
Solution Approach 2:
The patent replaces the mechanical disruption method (cell lysis) with a biochemical approach (enzymatic reverse transcription within intact cells). This substitution maintains cell integrity while achieving RNA-to-cDNA conversion through biochemical reactions that occur within the living cell environment.
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 rapid, scalable, and unbiased RNA sequencing at low cost, allowing for high-throughput analysis of gene expression and drug screening without the need for RNA isolation, maintaining cell integrity for further analysis.
Implementation Method 1
reverse transcription primers hybridizing to cellular RNA
Implementation Method 2
performing reverse transcription (RT) in situ within the cells
Implementation Method 3
using RT primers comprising a PCR handle 1, and partially biotinylated dNTPs
Implementation Method 4
releasing single stranded cDNA from the cells using a release mix, wherein the release mix comprises an RNAse
Implementation Method 5
introducing an adapter molecule comprising a PCR handle 2 that will bind 3' of the extended cDNA
Data Source
Figure 1A~1C
Figure 2
Figure 3A~3C
AI summary
The present invention relates to the field of biotechnology and a method for creating a cDNA library. More specifically, the invention refers to a method of forming complementary DNA (cDNA) sequencing libraries from RNA in situ comprising the steps of: (a) fixating cells, immobilized on a solid surface; (b) performing an in situ reverse transcription (RT) inside cells, using RT primers comprising a PCR handle 1, and partially biotinylated dNTPs; (c) releasing single stranded cDNA from the cells using a release mix, wherein the release mix comprises an RNAse, such that the single stranded cDNA is released from intact cells; (d) collecting a supernatant comprising released cDNA into a single larger volume or in separate volumes; and (e) introducing an adapter molecule comprising a PCR handle 2 that will bind 3' of the extended cDNA.