Fixed-Sample Nucleic Acid Detection with TSO and Randomer Primers
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Solution Overview
Problem
Existing methods for spatial transcriptomics struggle with degraded nucleic acids in fixed biological samples, such as FFPE samples, making it difficult to capture and analyze mRNA for gene expression data.
Innovation Solution
A method using a combination of template switching oligonucleotides (TSOs) and randomer primers during second strand synthesis to enhance the detection of nucleic acids from fixed biological samples, particularly FFPE samples, by generating multiple second strand synthesis products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If direct mRNA capture methods are used on fixed biological samples, then spatial transcriptomics data can be obtained, but the degraded mRNA quality in fixed samples makes detection difficult and reduces reliability
Solution Approach 1:
The patent introduces template switching oligonucleotides (TSO) as intermediary molecules that mediate between the degraded mRNA and the detection system. The TSO contains a template switching sequence that can bind to the 3' end of mRNA fragments, enabling second-strand synthesis even when the mRNA is degraded. This intermediary approach allows reliable detection of spatial transcriptomics data from fixed samples without requiring intact mRNA.
2Reliability
If fresh or fresh frozen samples are used for direct mRNA capture, then high quality mRNA detection is achieved, but the samples cannot be preserved for long-term storage and evaluation
Solution Approach 1:
The patent converts the harmful effect of formalin fixation, which normally degrades mRNA, into a beneficial preservation method. By using TSO-mediated second-strand synthesis, the method enables reliable detection of mRNA from formalin-fixed paraffin-embedded (FFPE) samples, thereby converting the harm of fixation-induced degradation into the benefit of long-term sample preservation with retained analytical utility.
3Productivity
If only a single primer is used during second strand synthesis, then the process is simple, but detection sensitivity and quantity of captured nucleic acids are reduced
Solution Approach 1:
The patent segments the primer function into two distinct components: a template switching oligonucleotide (TSO) with a template switching sequence that binds to the 3' end of mRNA, and a second primer that initiates second-strand synthesis. This segmentation allows each primer component to perform its specific function optimally, thereby enhancing detection sensitivity and the quantity of captured nucleic acids while maintaining a manageable level of complexity.
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 analysis of degraded mRNA from fixed samples, providing spatial transcriptomics data comparable to methods designed for fresh samples, thereby improving the usability of FFPE samples in gene expression studies.
Implementation Method 1
hybridizing the plurality of nucleic acids to the plurality of capture probes
Implementation Method 2
generating a population of extended capture probes by reverse transcription
Data Source
AI summary
Provided herein are method for determining a location of nucleic acids in fixed biological samples, in which the method includes use of a template switching oligonucleotide (TSO) and a randomer during second strand synthesis.