Mitigation effect of pfa-based tissue pretreatment method on spatiotemporal capture and diffusion
By immobilizing and decrosslinking paraformaldehyde, combined with sucrose concentration gradient replacement and OCT embedding, the problem of tissue diffusion in spatial transcriptome sequencing was solved, enabling in situ capture and accurate typing of mRNA.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- STOMICS TECH CO LTD
- Filing Date
- 2025-01-27
- Publication Date
- 2026-07-30
AI Technical Summary
During spatial transcriptome sequencing, tissue diffusion issues prevent accurate RNA capture, especially under conditions where fresh frozen embedding is difficult or unsuitable, resulting in poor sample testing results.
Samples were pretreated with paraformaldehyde fixative to cross-link free amino groups within the tissue to form a network structure. The network structure was then opened by appropriate decross-linking operations to fix RNA. Combined with sucrose concentration gradient replacement and OCT embedding, tissue diffusion was improved.
It effectively alleviates the problem of tissue diffusion, achieves in-situ capture and accurate typing of mRNA, and improves the accuracy of cellular mRNA expression mapping.
Smart Images
Figure PCTCN2025075512-FTAPPB-I100001 
Figure PCTCN2025075512-FTAPPB-I100002 
Figure PCTCN2025075512-FTAPPB-I100003
Abstract
Description
The mitigation effect of PFA-based tissue pretreatment on spatiotemporal capture diffusion Technical Field
[0001] This invention belongs to the field of space transcriptome sequencing. Specifically, this invention provides a sample processing method that, through sample pretreatment combined with subsequent decrosslinking steps, effectively alleviates problems such as tissue diffusion or untestable samples due to various reasons during space transcriptome sequencing, achieving, for example, in-situ capture of mRNA from tissue sections. Background Technology
[0002] Current technology involves directly cryopreserving and embedding samples without pretreatment. Frozen sections are then mounted for RNA capture within the tissue. Because the samples are not pre-fixed, the RNA remains in a free state during capture, making it more susceptible to diffusion problems.
[0003] Tissue diffusion is a widespread problem in space transcriptome sequencing technology. 10x Genomics Visium space transcriptome sequencing is currently the most widely used space omics technology and is commercially available. However, it still inevitably suffers from tissue diffusion. Furthermore, fresh cryopreservation has limitations, such as samples that cannot be freshly cryopreserved due to deformation, samples posing biosafety risks, lack of suitable conditions for fresh cryopreservation after sample collection, and low capture rates after testing fresh samples. Summary of the Invention
[0004] This invention uses paraformaldehyde as a fixative for sample pretreatment. Paraformaldehyde causes free amino groups in the tissue to cross-link and form a network structure, thereby fixing RNA in situ.
[0005] 1) This invention opens the internal network structure of tissues through appropriate decrosslinking operations, enabling RNA to be captured and improving the problem of tissue diffusion;
[0006] 2) Currently, some samples cannot be tested on the spatiotemporal transcriptome platform. PFA fixation pretreatment can improve or solve the above problems.
[0007] This invention relates to a novel tissue pretreatment method. Paraformaldehyde is used to pretreat samples, causing free amino groups within the tissue to cross-link and form a network structure, thus immobilizing RNA in situ. Then, through appropriate decross-linking operations, the network structure within the tissue is opened, allowing RNA to be captured and improving tissue diffusion. This invention not only provides a new pretreatment method for spatiotemporal transcriptomics technology but also enables better in-situ capture of mRNA. Through mRNA clustering analysis, accurate cell typing can be achieved, resulting in a more accurate real-time map of cellular mRNA expression.
[0008] This invention relates to the following aspects:
[0009] 1. A sample processing method for spatial transcriptome analysis, characterized by comprising the following steps: fixing the sample with paraformaldehyde, using sucrose to perform a concentration gradient displacement until the tissue state is a precipitated state, and embedding the sample.
[0010] 2. The sample processing method according to Project 1, characterized in that the concentration of paraformaldehyde is 1%-10%; or
[0011] The concentration of sucrose is 10%, 20%, and 30%, preferably with a volume ratio of sucrose solution to sample tissue greater than 10:1; or
[0012] The embedding method is OCT embedding.
[0013] 3. The sample processing method according to any one of items 1-2, characterized in that the tissue of the sample is fresh tissue or fixed tissue.
[0014] 4. The sample processing method according to any one of items 1-3, characterized in that the tissue of the sample is selected from the group consisting of: testis, skin, lung, fetal brain, spleen and small intestine.
[0015] 5. A method for spatial transcriptome analysis, characterized by comprising the following steps:
[0016] S1: Process the sample according to the sample processing method described in any one of items 1-4;
[0017] S2: The sample is brought into contact with the spatial array, and the oligonucleotide probes fixed on the spatial array are extended to obtain target nucleic acids with spatial information labels;
[0018] The oligonucleotide probe comprises a spatial sequence and a capture domain. The spatial sequence corresponds to the position of the oligonucleotide probe in the array, and the spatial sequence of the oligonucleotide probe at different positions is different. The capture domain is complementary to the target nucleic acid in the sample. The oligonucleotide probe is extended using the target nucleic acid as a template to obtain a spatially labeled target nucleic acid. The spatially labeled target nucleic acid includes the spatial sequence or its complementary sequence, and the target nucleic acid or its complementary sequence.
[0019] 6. The method according to Project 5, characterized in that:
[0020] In step S2, after the sample comes into contact with the spatial array and before the extension, the step pre-A is also included: fixing the sample with an organic solvent (preferably formaldehyde), and fixing the sample with an organic solvent (preferably formaldehyde) after de-crosslinking treatment.
[0021] Preferably, the decrosslinking includes decrosslinking with TE buffer at 60-95°C, more preferably at 65-95°C, 70-95°C or 75-95°C, and even more preferably at 90-95°C.
[0022] 7. The method according to Project 6, characterized in that, after the sample is fixed with an organic solvent and before decrosslinking, sample imaging is performed, preferably ssDNA staining imaging, immunofluorescence imaging, or HE staining imaging, preferably ssDNA staining imaging.
[0023] 8. The method according to Project 6, characterized in that, in the decrosslinking step, the pH of the TE buffer is 9-10, preferably 10.
[0024] 9. The method according to any one of items 6-8, characterized in that the decrosslinking is performed for 25-35 minutes, preferably 30 minutes.
[0025] 10. The method according to any one of items 6-9, characterized in that the method further comprises: performing tissue sectioning after step S1 and before step S2, and before fixing the sample with an organic solvent.
[0026] 11. The method according to any one of items 5-10, characterized in that, in step S2, after contact or fixation and before extension, the sample is permeated.
[0027] In a specific implementation plan, an exemplary process is shown in Figure 1.
[0028] The tissue pretreatment method provided by this invention is a spatiotemporal omics approach that can mitigate spatiotemporal capture diffusion and achieve in-situ capture of mRNA from tissue sections. Using a series of common raw materials (paraformaldehyde, sucrose, TE buffer, etc.), and through a simplified and refined process design, it effectively alleviates the spatiotemporal capture diffusion problem for some samples. It also provides a stable and effective solution for samples that cannot be freshly cryopreserved due to deformation, biosafety concerns, lack of suitable conditions for fresh cryopreservation after sampling, or low capture rates after fresh sample testing.
[0029] This method is low-cost, and with slight optimization of the original workflow of BGI's self-developed V1.2, it has excellent and stable results. It is applicable to the processing of various types of samples and has extremely broad application prospects, which can bring great economic benefits. Attached Figure Description
[0030] Figure 1. An exemplary flow of the method of the present invention.
[0031] Figure 2. Visual image of mouse testis control group (Bin10) (left) and cell image (right).
[0032] Figure 3. Visualized images of mouse testes (Bin10) (left) and cell images (right).
[0033] Figure 4. Visualized image of mouse skin control group (Bin10) (left) and cell image (right).
[0034] Figure 5. Visualized images of mouse skin test group (Bin10) (left) and cell images (right).
[0035] Figure 6. Visualized images of mouse lung control group (Bin10) (left) and cell images (right).
[0036] Figure 7. Visualized images of mouse lungs (Bin10) (left) and cell images (right).
[0037] Figure 8. Visual image of mouse fetal brain (Bin 50).
[0038] Figure 9. Visualization of fresh samples and PFA-fixed samples (Bin50).
[0039] Figure 10. Visualization of adjacent slices of the brain of a PFA-fixed mouse.
[0040] Figure 11. Visual images of adjacent sections of the testis of a PFA-fixed mouse.
[0041] Figure 12. Visual images of adjacent sections of skin from PFA-fixed mice.
[0042] Figure 13. Cluster consistency analysis of adjacent brain slices from PFA-fixed mice.
[0043] Figure 14. Cluster consistency analysis of adjacent testicular sections from PFA-fixed mice.
[0044] Figure 15. Cluster consistency analysis of adjacent skin sections from PFA-fixed mice. Detailed Implementation
[0045] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are conventional products that can be purchased commercially.
[0046] Experimental equipment: microtome (Leica), microscope (Mitoc PA53), Seq2000 sequencer (MGI)
[0047] Experimental materials: 4% paraformaldehyde (BOSTER), sucrose (BBI), PBS (10X) (GIBCO), SAKURA OCTCompound (SAKURA), Nuclease Free Water (NF water) (Ambion), 20X SSC (Ambion), TE buffer (Ambion), Sodium hydroxide solution (Aladdin), Methanol (Sigma), Qubit™ ssDNA Reagent (Invitrogen), STOmics Gene Expression Kit-S1 (BGI), STOmics Library Preparation Kit-S1 (BGI Genomics), Seq2000 Sequencing Kit PE100 (MBI)
[0048] Unless otherwise specified, the transcriptome analysis in the following embodiments was performed by the DCS Intelligent Cloud Platform (BGI, website: https: / / cloud.stomics.tech / ). However, there are various tools in the art that can perform spatial transcriptome analysis, and the above software or algorithms do not constitute a limitation on the scope of protection of this invention.
[0049] Example 1. Comparison of results between PFA fixed samples and fresh samples
[0050] Fresh samples of mouse testes, skin, and lung tissue taken within 30 minutes of ex vivo were used as materials.
[0051] Control group: The above materials were directly subjected to OCT cryo-embedding (without fixation).
[0052] Test group: Fresh samples taken within 30 minutes of in vitro examination were washed with 1xPBS and transferred to a container containing paraformaldehyde fixative. Fixation was performed at 4°C (fixation was complete when the tissue block settled to the bottom, e.g., 16-18 hours). The settled tissue block was then transferred to a 10% sucrose solution diluted with 1xPBS and purged at 4°C for 6-8 hours (fixation was complete when the tissue reached a precipitate state). The settled tissue block was then transferred to a 20% sucrose solution diluted with 1xPBS and purged at 4°C for 16-18 hours (fixation was complete when the tissue reached a precipitate state). The settled tissue block was then transferred to a 30% sucrose solution diluted with 1xPBS and purged at 4°C for 26-30 hours (fixation was complete when the tissue reached a precipitate state). The volume ratio of the solution to the tissue was greater than 10:1. During the sucrose solution purging process, the tissue could be gently agitated at any time intervals. If available, a shaker could be used to gently agitate the tissue to prevent deformation due to compression within the container.
[0053] During OCT embedding, dry ice or liquid nitrogen is used to rapidly freeze the sample to reduce ice crystal formation. Tissue sections of a certain thickness (5-15 μm) are attached to a chip containing single-stranded DNA probes. The tissue is then dried, fixed with organic solvents, and stained with DNA. After discarding the staining solution, the chip is washed with a low-salt solution, the wash solution is discarded, and the chip is dried by blowing or air drying. 100% glycerol is then added to the chip, and a coverslip is placed for photographing. The surface glycerol is then washed with a salt solution, and the chip is transferred to a pH 10 TE buffer at 60-95°C for 30 min to decrosslink. Finally, organic solvent fixation, permeabilization reagent addition, and tissue permeabilization are performed according to standard procedures.
[0054] After performing mRNA reverse transcription to obtain cDNA, the microarray is digested with enzymes to obtain cDNA with position probe information. This cDNA is then subjected to a series of subsequent operations, including library construction and sequencing. The sequence information of the mRNA with positional information is obtained, and the data is then analyzed to obtain in situ expression information of the mRNA or protein.
[0055] The reagents used in the above experiments were from the STOmics Gene Expression Kit-S1.
[0056] Chip preparation and sequencing were performed according to the standard procedures for DNB make and sequencing reagent preparation in MGI's Seq2000 sequencing kit PE100 (catalog number 1000013155). Data analysis was conducted using the Spatiotemporal Omics Visualization System (BGI).
[0057] result:
[0058] mouse testes
[0059] The testicular tissue of the control group mice was an untreated sample. RNA diffusion was observed in the seminiferous tubule cavity, making it impossible to accurately identify cell outlines. In dense areas, cells adhered together, making it impossible to distinguish single cells (Figure 2).
[0060] The testicular tissue of the test group mice was a sample that had been fixed and decrosslinked by PFA. There was no diffusion in the seminiferous tubule cavity, the gene capture was relatively uniform, the cell boundaries were clear, and the internal diffusion problem was alleviated (Figure 3).
[0061] mouse skin
[0062] The skin tissue of the control group mice was an untreated sample, and the visualization results showed severe diffusion, making it impossible to accurately identify cell outlines (Figure 4).
[0063] The skin tissue of the test group mice was a sample that had been fixed and decrosslinked by PFA. The visualization results showed clear outlines, which greatly improved the problem of tissue diffusion (Figure 5).
[0064] mouse lungs
[0065] The lung tissue of the control group mice was an untreated sample, and the visualization results showed severe diffusion, making it impossible to accurately identify cell outlines (Figure 6).
[0066] The lung tissue of the test group mice was a sample that had been fixed and decrosslinked by PFA. The visualization results showed clear outlines, which greatly improved the problem of tissue diffusion (Figure 7).
[0067] The results above show that the tissue pretreatment and subsequent decrosslinking procedures after PFA fixation in the test group were significantly better than those in the control group, especially for organs with severe diffusion as observed in the visualization results, greatly alleviating the diffusion problem. Therefore, at the level of tissue diffusion, the tissue pretreatment procedure after PFA fixation is superior to the procedure for fresh frozen samples.
[0068] Example 2. Sample processing when suitable conditions for fresh cryopreservation are unavailable.
[0069] Because fresh cryopreservation embedding of mouse fetal brain samples is not possible, the spatiotemporal transcriptome platform currently lacks data on such samples.
[0070] The steps of the test group in Example 1 were repeated using a fresh mouse fetal brain sample taken within 30 minutes of ex vivo in vitro.
[0071] Spatiotemporal omics analysis results:
[0072] The visualization results (Bin50) are shown in Figure 8.
[0073] See Table 1 for mRNA capture details:
[0074] Table 1. Mouse fetal brain mRNA capture status
[0075] The results above show that PFA pretreatment and decrosslinking treatment resulted in high gene capture and coverage that met the standards. PFA-fixed tissue pretreatment and decrosslinking treatment fill the gap in this type of tissue testing and solve the problem of not having suitable conditions for testing fresh cryopreserved samples.
[0076] Example 3. PFA tissue pretreatment method for improving low gene capture in fresh samples
[0077] Mouse spleen, mouse small intestine, and mouse lung (within 30 minutes of in vitro exposure) were used as materials. Some samples were directly used for gene capture, while others were processed by repeating the steps of the test group in Example 1. The effects of PFA tissue pretreatment and decrosslinking treatment on the low gene capture rate of fresh samples were evaluated.
[0078] Spatiotemporal omics analysis results:
[0079] Visualized image (Bin50): See Figure 9
[0080] The mRNA capture data is shown in Table 2:
[0081] Table 2. Comparison of mRNA capture between fresh samples and PFA-fixed samples
[0082] Based on the above results, some tissue genes in fresh samples showed low capture and varying degrees of tissue diffusion. Immobilized PFA and decrosslinked samples significantly improved capture and alleviated tissue diffusion problems.
[0083] Example 4. Stability of PFA tissue pretreatment results
[0084] Mouse brain, mouse testis, and mouse skin were extracted within 30 minutes of in vitro collection. Three adjacent tissue samples were taken from each tissue, and the experimental steps of the test group in Example 1 were repeated. Three adjacent tissue sections were obtained for each tissue. Data analysis and cluster consistency analysis were performed on each section.
[0085] Spatiotemporal omics analysis results:
[0086] See Figure 10-12 for the visualization image (Bin50).
[0087] See Table 3-5 for mRNA capture details:
[0088] Table 3. mRNA capture in adjacent brain slices of PFA-fixed mice
[0089] Table 4. mRNA capture data of adjacent testicular sections from PFA-fixed mice
[0090] Table 5. mRNA capture in adjacent skin sections of PFA-fixed mice
[0091] CV = (standard deviation / mean) × 100%. The CV values of adjacent slices are calculated using Bin200 Median MID and Bin200 Median Gene Type, respectively.
[0092] The results show that the gene count and MID CV values of three adjacent sections of the same tissue after PFA fixation and decrosslinking in the test group were both less than 15%, indicating high intragroup stability. The correlation coefficients among the three adjacent sections of the same tissue were also high (all above 0.97). The CV values indicate good correlation in gene expression and good cluster consistency. In conclusion, the PFA-fixed tissue pretreatment method demonstrates high stability.
Claims
1. A sample processing method for spatial transcriptome analysis, characterized in that... The process includes the following steps: fixing the sample with paraformaldehyde, using a concentration gradient of sucrose to replace the sample in a precipitated state, and then embedding the sample.
2. The sample processing method according to claim 1, characterized in that, The concentration of the paraformaldehyde is 1%-10%; or The concentration of sucrose is 10%, 20%, and 30%, preferably with a volume ratio of sucrose solution to sample tissue greater than 10:1; or The embedding method is OCT embedding.
3. The sample processing method according to any one of claims 1-2, characterized in that, The tissue in the sample is either fresh or fixed.
4. The sample processing method according to any one of claims 1-3, characterized in that, The tissues in the sample were selected from the group consisting of: testis, skin, lung, fetal brain, spleen, and small intestine.
5. A method for spatial transcriptome analysis, characterized in that... Includes the following steps: S1: Sample processing is performed according to the sample processing method described in any one of claims 1-4; S2: The sample is brought into contact with the spatial array, and the oligonucleotide probes fixed on the spatial array are extended to obtain target nucleic acids with spatial information labels; The oligonucleotide probe comprises a spatial sequence and a capture domain. The spatial sequence corresponds to the position of the oligonucleotide probe in the array, and the spatial sequence of the oligonucleotide probe at different positions is different. The capture domain is complementary to the target nucleic acid in the sample. The oligonucleotide probe is extended using the target nucleic acid as a template to obtain a spatially labeled target nucleic acid. The spatially labeled target nucleic acid includes the spatial sequence or its complementary sequence, and the target nucleic acid or its complementary sequence.
6. The method according to claim 5, characterized in that: In step S2, after the sample comes into contact with the spatial array and before the extension, the step pre-A is also included: fixing the sample with an organic solvent (preferably formaldehyde), and fixing the sample with an organic solvent (preferably formaldehyde) after de-crosslinking treatment. Preferably, the decrosslinking includes decrosslinking with TE buffer at 60-95°C, more preferably at 65-95°C, 70-95°C or 75-95°C, and even more preferably at 90-95°C.
7. The method according to claim 6, characterized in that, After the sample is fixed with an organic solvent and before decrosslinking, the sample is imaged. Preferably, the imaging is ssDNA staining imaging, immunofluorescence imaging, or HE staining imaging, with ssDNA staining imaging being the most preferred.
8. The method according to claim 6, characterized in that, In the decrosslinking step, the pH of the TE buffer is 9-10, preferably 10.
9. The method according to any one of claims 6-8, characterized in that, The decrosslinking process is carried out for 25-35 minutes, preferably 30 minutes.
10. The method according to any one of claims 6-9, characterized in that, The method further includes: performing tissue sectioning after step S1 and before step S2, and before fixing the sample with an organic solvent.
11. The method according to any one of claims 5-10, characterized in that, In step S2, after contact or fixation but before extension, the sample is permeated.