Spatial Transcriptomics Library Prep for FFPE mRNA Capture
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Formalin fixation and subsequent de-crosslinking in FFPE tissues cause degradation and chemical modification of RNAs, making poly-A capture of mRNA more challenging than in fresh frozen tissue, leading to incomplete transcriptomics libraries.
Innovation Solution
A method involving mounting tissue samples on a substrate with capture oligonucleotides, hybridizing gene-specific probes to mRNA transcripts, ligating them, and capturing ligated pairs to improve mRNA transcript capture efficiency, generating a more complete transcriptomics library.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If poly-A capture method is used on FFPE tissues, then mRNA can be captured, but capture efficiency is low due to RNA degradation and chemical modification
Solution Approach 1:
The capture method is divided into multiple sequential steps: initial poly-A capture, followed by template switching oligo addition, then second poly-A capture. This segmentation allows each step to optimize for specific functions, improving overall capture efficiency despite RNA degradation in FFPE tissues.
Solution Approach 2:
A template switching oligo is added to the 3' end of mRNA during the first reverse transcription step before the second poly-A capture. This preliminary action creates a known sequence handle that enables more efficient subsequent capture and library preparation, compensating for RNA degradation.
2Quantity of substance
If traditional poly-A capture is used, then mRNA can be isolated, but the transcriptomics library is incomplete
Solution Approach 1:
The template switching oligo acts as an intermediary element that bridges the first and second capture steps. It provides a known sequence that facilitates efficient second poly-A capture and enables comprehensive transcript recovery, reducing loss of mRNA transcripts and improving library completeness.
Solution Approach 2:
The method changes the capture parameters by performing two sequential poly-A captures with different conditions and adding template switching oligos. This parameter variation allows recovery of transcripts that would be missed by a single capture method, improving overall library completeness.
3Ease of operation
If de-crosslinking is performed on FFPE tissues, then RNA accessibility is improved, but RNA degradation increases
Solution Approach 1:
Reverse transcription with template switching oligo addition is performed as a preliminary action before second poly-A capture. This allows cDNA synthesis to protect RNA while simultaneously creating handles for subsequent capture, improving RNA accessibility without additional degradation from repeated de-crosslinking.
Solution Approach 2:
The method maintains continuous useful action by performing reverse transcription and capture in an integrated workflow without removing samples from the reaction environment. This continuity preserves RNA integrity while maintaining accessibility through the chemical environment already established for de-crosslinked FFPE tissues.
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
Enhances the efficiency of capturing mRNA transcripts from FFPE and fresh frozen tissues, resulting in a more complete transcriptomics library with improved genetic information for disease analysis.
Implementation Method 1
contacting the tissue sample with i) a plurality of 5' gene specific probes comprising a sequence complementary to the first universal adapter sequence and a 5' gene specific primer; and ii) a plurality of 3' gene specific probes comprising a 3' gene specific primer, a unique molecular index, and a second universal adapter sequence, under conditions such that one or more 5' gene specific probe and one or more 3' gene specific probe hybridizes to one or more mRNA transcript in the tissue sample
Implementation Method 2
contacting the tissue sample in (b) with ligation reagents such that a 5' gene specific probe and a 3' gene specific probe hybridized to the mRNA transcript in proximity to each other are ligated together to form one or more ligated gene specific probe pairs
Implementation Method 3
capturing the ligated gene specific probe pair oligonucleotide of (d) on the substrate by binding of the sequence complementary to the first universal adapter sequence in the 5' gene specific probe to the first universal adapter sequence of the capture oligonucleotide
Data Source
AI summary
The present disclosure relates, in general, to methods for improving preparation of a spatial transcriptomics RNA, library, for example a mRNA library, by improving capture of RNA transcript information from a tissue sample in situ. The spatial transcriptomics library from a tissue sample is useful to determine a genetic profile and help diagnose a person who has or is at risk of having a disease, such as cancer, genetic disease, autoimmune disease, and other indications, and improve treatment of the subject.