Oligonucleotide Primer Pool Design for rRNA Exclusion
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Solution Overview
Problem
Current reverse transcription methods using oligo dT and random primers face limitations, such as excessive amplification of rRNA-derived products leading to background signals in microarray hybridizations, and inability to efficiently transcribe non-coding RNAs and partially degraded RNAs, while oligo dT primers introduce 3' bias and are unsuitable for all RNA types.
Innovation Solution
Designing a pool of oligonucleotides that do not substantially hybridize to unwanted sequences, using computational and physical selection methods to create a pool of oligonucleotides with non-identical hybridization sequences that effectively avoid rRNA sequences, allowing for efficient reverse transcription of most RNAs without rRNA contamination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If random primers are used for reverse transcription, then all RNA molecules can be transcribed, but rRNA-derived products dominate the cDNA population causing high background signals
Solution Approach 1:
The patent extracts and removes rRNA sequences from the transcriptome database before designing random primers. This ensures that the generated primer pool does not contain sequences that would hybridize to rRNA, thereby eliminating the source of background signal while preserving coverage of other RNA species.
Solution Approach 2:
The patent introduces an intermediary computational filtering step that processes the transcriptome database to exclude rRNA sequences. This intermediary process acts as a mediator between the random priming approach and the final cDNA synthesis, preventing rRNA amplification without requiring physical removal of rRNA from the sample.
2Quantity of substance
If oligo dT primers are used, then mRNA enrichment is achieved, but 3' bias is introduced and full-length cDNA synthesis is limited
Solution Approach 1:
The patent segments the priming strategy into two components: (1) oligo dT primers for mRNA enrichment at the 3' end, and (2) computational random primers designed to cover the entire transcriptome including 5' regions. This segmentation allows simultaneous achievement of mRNA enrichment and full-length coverage.
Solution Approach 2:
The patent merges oligo dT priming with computational random priming in a single reverse transcription reaction. The computational random primers are designed to be complementary to various positions across all transcripts, and when combined with oligo dT, provide both 3' enrichment and comprehensive full-length coverage.
3Object-affected harmful factors
If rRNA removal steps are added, then background signal is reduced, but experimental complexity and cost increase
Solution Approach 1:
The patent performs preliminary action by computationally removing rRNA sequences from the transcriptome database before primer design. This preventive measure eliminates the need for subsequent physical rRNA removal steps, as the primers are already designed to avoid rRNA sequences.
Solution Approach 2:
The patent creates a computational copy of the transcriptome database and processes this copy to exclude rRNA sequences. This virtual filtering allows primer design that inherently avoids rRNA without requiring physical manipulation or removal of rRNA from the actual RNA sample.
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 reduces rRNA-derived cDNA contamination, enhances detection sensitivity, and provides full-transcript coverage without introducing 3' bias, enabling better analysis of the entire transcriptome, including non-coding RNAs and degraded samples.
Implementation Method 1
employing a selection method to determine which hybridization sequences of length n are not expected to substantially hybridize to the exclusion sequence
Data Source
AI summary
The present invention provides novel algorithms for designing oligonucleotides that do not substantially hybridize to a small group of unwanted transcripts, while hybridizing to most other transcripts. Such oligonucleotides are particularly useful as primers for reverse transcription. The invention also provides compositions containing oligonucleotides that do not substantially hybridize to a small group of unwanted transcripts, while hybridizing to most other transcripts.