Question: Is bone compatible with Xenium In Situ Gene Expression?
Answer: Below is a summary of our learnings and considerations from working with healthy mouse femur, tibia, sternum, and neonatal mouse bone, as well as human tibia, femur, and bone marrow for Xenium. We cannot guarantee assay performance even when implementing the guidance listed in this article and therefore, customers can proceed at their own risk. 10x Genomics Support does not have additional information beyond what is provided in this article and will not be able to provide additional details from internal experiments or guidance.
Histologically & spatially speaking, bone is notorious for being a sample that has a high rate of tissue detachment and typically being known as RNA-poor. Data quality is very much dependent on the organism (mouse vs. human), the type of bone, the starting sample quality, and tissue processing.
Before deciding which sample preparation method is most appropriate for the experimental design, it is important to determine the following:
- Does the sample of interest contain any calcified tissue remaining or whether it is soft tissue only?
- Is the organism human or mouse?
If the sample (human or mouse) does not contain any calcified tissue please refer to 'Is bone-derived soft tissue compatible with Xenium In Situ Gene Expression?’.
If the sample contains calcified tissue, it is important to decalcify the tissue prior to sectioning. Residual calcified bone can damage surrounding tissue and blades during section and will not yield good Xenium data. The methods for decalcification vary depending on the organism of interest. Decalcification is a delicate balance of gaining access to RNA by removing hard bone, while avoiding RNA damage that is inherent to the method as the chemicals generally degrade RNA, impacting the RNA in marrow more severely due to mismatch of prep methods. Multiple decalcification methods were tested to remove hard bone. For mouse, we found EDTA, EDTA/PFA, and formic acid to perform similarly. For human, we found EDTA or EDTA/PFA to be the most effective method, striking a balance between efficient decalcification while preserving RNA quality, thus ensuring optimal Xenium results.
Mouse Protocol Overview:
Freshly collected bone from adult C57bl/6 mice:
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Cardiac perfusion with 4% PFA.
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Thermo Scientific (AAJ19943K2); Thermo Scientific (AA433689L, requires dilution to 4%)
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Thermo Scientific (AAJ19943K2); Thermo Scientific (AA433689L, requires dilution to 4%)
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Tissue fixation: 10% NBF at 4°C O/N (18-24h) fixation.
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Decalcification:
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Dehydration:
- 1xPBS wash (3x).
- Dehydration with 30%, 50% and 70% EtOH 45min-1h each step (store in 70% EtOH at -20°C if needed).
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Processing:
- Put into cassettes.
- Processing default O/N protocol (Using Epredia Excelsior).
- Embed in paraffin in plane ideal for sectioning hard bone & bone marrow.
Tissue Fixation (detailed methods):
- Bones were collected fresh from adult C57bl/6 mice.
- Cardiac perfusion with 4% PFA was performed on mice.
- Tissue was then fixed in 10% NBF at 4°C O/N (18-24h) fixation.
- Optimal fixation time will be variable depending on the composition and size of the sample.
- Tissue should be fixed with fresh formaldehyde. Fixative penetration is key, generate a coronal or axial section to achieve more consistent fixation.
- Additional 10x Tips and Tricks for fixation are listed here:
- General recommendations and best practices can be found in this Leica article: An Introduction to Specimen Processing.
Decalcification (detailed methods):
Decalcification is required to perform histological, immunological, and molecular studies on bone tissue, but a consequence of decalcification is RNA fragmentation. Thus, optimizing conditions that maximize bone softening while retaining RNA integrity is critical for performing molecular experiments with bone. Decalcification can be performed on the surface of an FFPE block or it can be performed during or after fixation, prior to embedding. In these studies, we tested decalcification immediately after fixation and thus, other decalcification methods will require additional optimization. We tested EDTA-based decalcification methods as well as acid decalcification methods (e.g., formic acids) [1,2]. Other acid decalcification methods were not tested internally (e.g. hydrochloric, and nitric acid).
EDTA recommendations based on limited testing:
- Prepare 0.5M EDTA (PH 8.0).
- Tip: EDTA powder will not dissolve in more acidic solutions, ensure that the solution is at pH 8 so EDTA is fully in solution
- Utilize nuclease-free reagents so as to not introduce nucleases
- Ensure to remove as much muscle and soft tissue as possible as it will impinge on the decalcification process.
- Fully submerge tissue in a sufficient volume of EDTA solution and utilize a stir bar or nutator to gently agitate the solution during decalcification.
- Perform decalcification with 0.5M EDTA (PH 8.0) for ~3 days at room temperature or 4°C.
- Time for decalcification depends on bone thickness. A time course will be required for optimization; probing the tissue to determine whether bone is still rigid.
- Decalcification is complete once the bone is pliable (as determined by gently poking the tissue with sharp metal forceps).
- Wash the decalcified bone tissue several times with 1x PBS pH 7.4 (3x), then proceed to processing and embedding.
Formic acid recommendations based on limited testing:
- Formic acid was prepared by diluting Sigma (PHR3559) to 10% in DI water.
- Decalcification was performed with 10% formic acid ~24 hours at room temperature or 4C.
- Like EDTA-based decalcification methods, bone rigidity was tested with a probe to avoid under- or over- processing.
- Wash the decalcified bone tissue several times with 1x PBS pH 7.4 (3x), then proceed to processing and embedding.
Processing and Embedding:
- To minimize RNA fragmentation, use low-melting paraffin wax for embedding.
- General recommendations and best practices can be found in this Leica article: An Introduction to Specimen Processing.
Block Selection:
- During sectioning, if there is evidence of insufficient decalcification (e.g., sectioning is difficult), it may be necessary to select a different block (see below for additional information).
Sectioning:
- Ensure to clean the workstation, microtome, tools, and blades with RNase Decontamination Solution before sectioning to minimize introducing exogenous RNases.
- Utilize a clean, new blade for sectioning
- 5 µm section thickness has been validated for FFPE samples going into Xenium In Situ Gene Expression.
- During testing, we utilized 5 µm sections with bone samples
- If the blade is chattering during sectioning or the block is too hard to generate a good quality section, the block may not be efficiently decalcified.
- Consider warming the block using lukewarm water (37°C) to soften and hydrate it prior to sectioning
- Similar protocols suggest performing surface decalcification and resectioning, but this method has not been validated with this assay
- After tissue sections have been placed, tissues should be dried as per the recommendations in the Xenium In Situ for FFPE - Tissue Preparation Guide and then stored in a room temperature desiccator until ready to proceed to the staining protocol
Sample preparation:
- No modifications were made to Xenium In Situ for FFPE Tissues – Deparaffinization & Decrosslinking.
- As bone tissue is more prone to detachment, it is important to follow best practices.
- Ensure to follow recommended drying steps outlined in the Tissue Preparation Guide.
- During deparaffinization and decrosslinking, exercise caution in immersion steps so as to not disrupt the tissue.
- When pipetting in Xenium Slide Cassettes, dispense and remove reagents along the side of the well without touching or pipetting on top of the tissue.
Panel selection:
- The Mouse Tissue Atlassing Panel can be used with bone, but we recommend an add-on panel to maximize bone/bone marrow-specific cell types and genes. For example, for data highlights, Osteoblasts, Osteocytes, Osteoclasts, Chondrocytes were targeted with a 100 gene add-on panel to increase specificity.
User Guide:
- Follow the User Guide as written for the panel plexy desired. All Xenium In Situ User Guides can be found here.
Mouse Femur Data Highlight:
Preview dataset of mouse femur decalcified according to methods outlined here and run with the Xenium In Situ Gene Expression Assay and the Xenium Mouse Tissue Atlassing Panel.
- The Xenium dataset generated by this experiment was good quality, as nuclear decoded transcripts per 100 µm2 were high (>200 µm2 ). Furthermore, 68 median transcripts per cell were identified.
- In total, ~256K cells were detected and over 24 million high quality decoded transcripts.
Human Protocol Overview:
A proof of concept was performed on freshly collected human bone from the femoral head. Tip: Keep some surrounding muscles to help cortical bone anchor to slides.
- Tissue fixation: 10% NBF at 4°C O/N (18-24h) fixation.
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Decalcification:
- 1xPBS wash (3x).
- Decalcification:
- 15% 0.5M EDTA (PH 8.0)/0.4% PFA for ~3-4 weeks RT or 4°C.
- Tips: Use probe to determine if bone is less rigid (more gelatinous). Change to fresh EDTA solution every 2-3 days; agitate sample gently.
- Human bone may take months for optimal decalcification depending on the size and bone region.
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Dehydration:
- 1xPBS wash (3x).
- Dehydration with 30%, 50% and 70% EtOH 45min-1h each step (store in 70% EtOH at -20°C if needed).
-
Processing:
- Put into cassettes.
- Processing default O/N protocol (Using Epredia Excelsior).
- Embed in paraffin in plane ideal for sectioning hard bone & bone marrow.
Human Tibia Data Highlights:
Preview dataset of human femur decalcified according to methods outlined here and run with Xenium In Situ Gene Expression Assay and the Xenium Human Multi-Tissue and Cancer Panel.
- We observe 27 Median Transcripts per Cell across the entire bone tissue which includes both calcified bone (RNA-poor) and also marrow (RNA-rich).
- Despite decalcification, nuclear transcript density was high at 76.2 nuclear transcripts per 100 um2.
- Transcript density varies with morphology - high density in marrow-rich regions and low density in calcified regions.
- Taken together, these results demonstrate the current decalcification protocol preserves tissue integrity and RNA in both calcified and soft tissue regions.
References:
- Sighn, V. M., et al., “Analysis of the effect of various decalcification agents on the quantity and quality of nucleic acid (DNA and RNA) recovered from bone biopsies.” Ann Diagn Pathol. 2013; 17(4):322-6. DOI: 10.1016/j.anndiagpath.2013.02.001
- Zheng, G., et al., “Clinical Mutational Profiling of Bone Metastases of Lung and Colon Carcinoma and Malignant Melanoma Using Next-Generation Sequencing.” Cancer Cytopathol. 2016;124:744-53. DOI: 10.1002/cncy.21743
Product: Xenium In Situ Gene Expression