Unified cDNA Library Preparation for Mixed RNA Samples
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Solution Overview
Problem
Conventional methods for sequencing total RNA samples are cumbersome as they require separate workflows for mRNA and small RNAs, making it inefficient to process RNA samples containing a mix of RNA molecules varying in length.
Innovation Solution
A method is developed to create a cDNA library by reverse transcribing RNA samples, treating the products with RNAseH to produce mRNA fragments, and then reverse transcribing these fragments, allowing for the inclusion of both mRNA and small RNAs in a single workflow, enabling the production of a cDNA library that contains cDNA copies of both mRNA fragments and small RNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If separate workflows are used for mRNA and small RNA sequencing, then each RNA type can be processed with dedicated protocols, but the overall process becomes cumbersome and inefficient
Solution Approach 1:
The patent merges the separate mRNA and small RNA sequencing workflows into a single unified cDNA library preparation protocol. By using a common reverse transcription step with oligo(dT) priming followed by RNase H treatment and second-strand synthesis, both RNA types are converted to cDNA and sequenced together, eliminating the need for parallel processing while maintaining sequencing quality
Solution Approach 2:
The patent creates a universal cDNA library preparation method that can process both mRNA and small RNA molecules through the same protocol. The methodology uses universal reagents and conditions (reverse transcriptase, RNase H, DNA polymerase) that work for both RNA types, making the system multi-functional and applicable to diverse RNA species in a single workflow
2Productivity
If a single workflow is used for both mRNA and small RNA, then processing efficiency is improved, but the complexity of handling different RNA types increases
Solution Approach 1:
The patent employs parameter changes to optimize the unified workflow for handling both RNA types. By adjusting incubation temperatures, enzyme concentrations, and reaction times during reverse transcription and cDNA synthesis, the protocol accommodates the different size and structural characteristics of mRNA and small RNA molecules, enabling efficient processing without requiring separate optimization for each RNA type
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 streamlines the sequencing process by integrating the handling of mRNA and small RNA fragments into a single cDNA library, improving efficiency and reducing the complexity of workflows for RNA sample analysis.
Implementation Method 1
reverse transcribing an RNA sample that comprises mRNA to produce a first strand cDNA product
Implementation Method 2
treating the first strand cDNA product with RNAseH to produce mRNA fragments
Implementation Method 3
reverse transcribing the small RNAs and the mRNA fragments to produce a cDNA library
Data Source
AI summary
This disclosure provides, among other things, a method for making a cDNA library. In some embodiments the method may comprise reverse transcribing mRNA to produce DNA:mRNA hybrids, treating the DNA:mRNA hybrids with RNAseH to produce mRNA fragments, and reverse transcribing the mRNA fragments.