InquirySpatial transcriptome technologies not only provide information on transcriptome data, but also localize spatial locations in tissues, which are important for research in many fields such as cancer pathogenesis, neuroscience, and developmental biology.
Traditional spatial transcriptome sequencing techniques are limited to fresh frozen tissue sections and cannot be more effectively applied to FFPE (formalin-fixed paraffin-embedded) samples, which are commonly used in clinical practice. 10X Genomics launched a new Visium spatial gene expression solution for FFPE samples, which enables the acquisition of highly sensitive and specific gene expression information and spatial location information from FFPE samples. Visium FFPE Sample Gene Expression is a pioneering solution that complements traditional pathology analysis methods and further expands the possibilities of spatial biology research.
Technical Principle
Visium FFPE Spatial Gene Expression is based on the principle of probe hybridization to obtain tissue spatial gene expression maps from FFPE tissue samples, and realize true in situ detection under the background of morphology.
In this technology, FFPE tissue sections are placed on the Visium gene chip for histomorphological staining and imaging, and then the probes on the Visium gene chip are used for quantitative expression. Due to the high degree of RNA degradation in FFPE samples, the Visium Spatial Gene Expression Solution for FFPE samples cannot utilize the poly(dA) sequence of intact mRNA for mRNA capture like fresh frozen tissue samples. This protocol uses paired RNA-templated ligation probes (RTL) targeting the coding gene to hybridize to mRNA and then ligate to each other, while the mRNA is degraded by RNase. Through tissue permeabilization, the connected paired probes are captured with primer sequences containing Barcode, UMI and poly(dT) sequences on the chip, and then the probe sequences carrying the space Barcode are used for library construction and sequencing to obtain gene expression information and spatial location information.
Probe capture
Principle of spatial transcriptome sequencing of FFPE samples (10xgenomics.com)
Advantage
1. Allow more efficient use of the large number of FFPE samples in clinical settings.
2. RNA template ligation (RTL) probe set with high sensitivity and specificity and a probe set covering over 18,000 genes in human and mouse.
3. Can combine a wider range of pathological and prognostic information to screen for biomarkers, etc.
a. Reuse of preserved or biospecimen library samples to discover biomarkers that can be studied retrospectively and retrospectively;
c. Combine with immunofluorescence techniques to visualize protein and gene expression, or combine with HE staining for morphological studies;
d. Entire FFPE sections can be studied without missing important biological information.
Sample Requirements
Human, mouse FFPE samples.
1. Meet the requirements for tissue patch experiments (no desquamation).
2. RNA integrity: DV200>50%.
Workflow
FFPE transcriptome transcriptome sequencing workflow (10xgenomics.com)
Analysis Process
Q&A
Q1: Besides human and mouse FFPE samples, do you support spatial transcriptome sequencing of FFPE samples from other species?
A: No, it does not. Currently, FFPE samples only support spatial transcriptome sequencing for human and mouse, and the Visium FFPE spatial gene expression solution designs a pair of capture probes for targeting human and mouse genes, covering 18,000 genes for human and 20,000 genes for mouse.
Q2: Does Visium analysis require tissue optimization?
A: The Visium spatial gene expression solution for FFPE does not require tissue optimization. However, the Visium spatial gene expression solution for fresh frozen samples requires tissue optimization due to the differences in chemistry and molecular biology between the two sample preparation processes.
Q3: Are the data from spatial transcriptome sequencing of FFPE samples reliable compared to fresh frozen tissue samples?
A: Officially, 10x Genomics has tested the Visium analysis of mouse brain FFPE and fresh frozen tissue separately and found that the genes detected in the two tissues are highly correlated, demonstrating the reliability and high sensitivity of the Visium FFPE spatial gene expression solution.
Reference
- Tertiary lymphoid structures generate and propagate anti-tumor antibody-producing plasma cells in renal cell cancer.
