Q1: Is it possible to perform spatial transcriptome analysis on plant tissues?

In theory it is possible. Plants can also be captured by the polyA structure of mRNA.

However, analysis of mature tissues is not recommended.

There are two main reasons: first, the vacuoles of mature tissues are large, so that the number of cells in a single detected spot is extremely low, and the gene expression activity is not very high, which is not conducive to the subsequent data presentation;

Second, mature plants, such as mature stems, have a large number of vessels and sieve tubes, the former are basically dead cells, and the latter have basically no nuclei and few gene expression.

The cell density and data quality of primary tissue will be higher than that, so if you want to do the spatial transcriptome of plants, it is more recommended to use primary tissue samples.

Q2: Will there be cross-contamination between Spots? How is diffuse cross-contamination assessed?

According to the current project experience, when the permeabilization time is selected properly, the captured mRNA will not cross-link, and there will be no cross-contamination between spots. This can be demonstrated in two ways:

(1) When doing tissue optimization, we can visualize the capture of mRNA through fluorescent reverse transcription. If diffusion occurs, cross-contamination will definitely occur during permeabilization at this time;

(2) Evaluate based on the expression of specific marker genes. For example, if a gene expressed only in the hippocampus is also expressed in the surrounding tissues of the hippocampus, it means that cross-contamination has occurred.

Frozen Embedding

Q3: Can only methanol be used as the fixative?

At present, it is more recommended to use methanol fixation. The principle of methanol fixation is dehydration, which will not cause too much chemical reaction. As for formaldehyde, the principle of fixation is protein cross-linking, which may cause cross-linking of RNA and protein and affect the subsequent RNA release and reduce the data quality.

Q4: How to determine the best time for tissue permeabilization?

It can be determined by using a tissue optimization chip, permeabilizing the tissue according to the gradient time, and selecting the best time according to the permeabilization situation. The selection criteria are as follows:

Select the time when the fluorescent signal is the strongest;

When the fluorescent signal is similar, choose the time with the smallest diffusion (indicating that there is no cross-contamination of mRNA);

When the fluorescence signal intensity and diffusion conditions are the same, select a longer time point (to ensure the release of as much mRNA as possible).

Q5: How to choose the frozen embedding method?

There are two main strategies for cryoembedding: freezing first and then embedding, and freezing and embedding at the same time.

Method 1: Freezing followed by embedding

Tissues were snap-frozen in isoamyl alcohol cooled with liquid nitrogen, and then OCT solidification and tissue embedding were completed in dry ice.

Method 2: Simultaneous freezing and embedding

Submerge the tissue in OCT, place it on dry ice, and complete tissue freezing and embedding simultaneously with cooling.

Q6: Advantages and disadvantages of two freeze-embedding method

A6: Both of above two approaches have pros and cons:

The first method can quickly cool down and protect the integrity of cells, but exposure to relatively high temperature OCT during embedding may cause cell lysis at the edge of the tissue;

The second method does not cause secondary freezing and thawing of the tissue, and the overall integrity of the cut surface is better. However, the operation process is slightly complicated, and it is not easy to operate on the operating table or when sampling outside.

According to the current project experience, the second dry ice embedding method is suitable for more tissue types and can obtain better sectioning results. However, it still depends on the type of organization. For some smaller and lighter samples such as puncture samples, it is generally recommended to use liquid nitrogen combined with isopentane for frozen embedding.

There are also certain differences in the adaptability of different tissue types, so it is best to do a pre-experiment and then make a selection based on the results of the pre-experiment.

Q7: Can I use a sample that has been stored in liquid nitrogen for a long time for spatial transcriptome?

A7 Liquid nitrogen freezing will produce ice crystals on the surface of the tissue, and the ice crystals will affect the tissue structure of the section. There may be degradation of RNA when stored for a long time. In this case, if the sample can be reacquired, it is better to re-prepare with pre-cooled isopentane embedding. If the pre-collected samples cannot be re-obtained, sample sectioning can be attempted. Generally, 10 sections are first sliced for RNA quality control test, and if the quality control is passed, the sections can be stained with HE. If the staining results show that the tissue structure of the region of interest is intact, then subsequent experiments can be performed. If the structure is severely damaged, then subsequent experiments cannot be performed even if the RNA quality is intact.

Paraffin Embedding

Q8: How long can paraffin-embedded samples be stored?

A8: There are many factors that degrade RNA during the process of embedding and storage, such as formaldehyde fixation, high temperature wax immersion, tissue oxidation, etc. Depending on the time and storage conditions, the rate of RNA degradation will be different. Therefore, it is inaccurate to judge whether paraffin-embedded samples are usable based on storage time alone. At present, it is mainly based on the RNA DV200 (percentage of RNA with fragment length greater than 200nt) in the embedded sections, and it can be used when it reaches 50%.

Q9: Is it possible to make FFPE sections for spatial transcriptome for any species?

A9: This includes the 10x Visium technique as well as the DSP histology technique, both of which are currently only available for human and mouse. The data is captured by probes, and different species use different probe systems, so all other species are not available for now.

Q10: What are the advantages of paraffin embedding compared to freeze embedding?

A10: First, paraffin-embedded samples can be stored for a long time, and retrospective research can be performed on the samples later. However, frozen sections of long-term frozen tissues may destroy the structure of tissue cells due to the formation of ice crystals, resulting in diffusion of antigens and less accurate localization.

The second is that it can make up for the disadvantages of poor freezing and embedding of some tissues. If the permeabilization effect is poor or the tissue is highly soluble, paraffin embedding can be used. At the same time, paraffin sections can be cut into thinner sections, and in situ hybridization probes can easily penetrate into the tissue, and the color and shape of the obtained data will be better than frozen sections.

Sample

Q11: Which samples can be used for spatial transcriptome analysis

A11: Human: brain (cerebellum, cerebral cortex, glioblastoma multiforme), spinal cord (normal), heart (normal and myocardial infarction), kidney (normal and nephritis), colon (colorectal cancer), lung (normal, papillary adenocarcinoma) ovary (tumor), breast (normal, invasive ductal carcinoma, invasive lobular carcinoma), spleen (inflammation), lymph (normal, inflammation), etc.

Mice: brain, heart, small and large intestine, stomach, liver, kidney, quadriceps, lungs, testes, thyroid, thymus, eyes, tongue, olfactory bulb, spleen, etc.

Rats: brain, kidney, heart, olfactory bulb, etc.

Q12. Are there any requirements for the size of the tissue in the spatial transcriptome?

A12: The capture area of the Spatial Transcriptomics Visium chip is 6.5mmX6.5mm, so before selecting samples, first confirm the best sampling location and reasonable tissue size to ensure the effective utilization of the Visium chip. Therefore, it is best if the sample size and capture area are more appropriate. Samples larger than the area of the capture area can be considered to be placed in two capture areas. For samples smaller than the area of the capture area, it is difficult to put multiple samples in one area, so it only can be try to do experiments.

Q13. Is there any limit to the number of samples in the spatial transcriptome, and must the number of samples be a multiple of 4?

A13: One slide has four capture areas. In the spatial transcriptome experiment, the four capture areas of a slide must be operated together, and one slide will still be consumed if less than four samples are filled, so in terms of cost, 4 samples or multiples of 4 samples is the optimal solution

tissue permeabilization

Q14: The spatial transcriptome needs to be pre-permeabilized in the early stage. Will the optimal conditions not be found during pre-permeabilization?

A14: For pre-permeabilization, we have tested many samples. The optimal permeabilization time of the sample is actually a relatively long time range, within which a good mRNA release effect can be achieved. Therefore, general tissues can basically find the best permeabilization time through pre-experimental exploration.

Q15. As mentioned above, it is best to analyze four samples at a time for spatial transcriptome, so does each sample need to be pre-permeabilized separately?

A15: Normally, the optimal permeabilization time for normal samples is almost the same, so if the four samples are normal tissue samples, but the processing is slightly different, you can choose one sample for pre-permeabilization; but for samples in a disease state, the optimal permeabilization time will vary greatly. It is recommended to conduct a pre-permeabilization experiment for each sample to explore the optimal permeabilization time.

Other

Q16 Is the detection of spatial transcriptome at the single-cell level?

A16 The detection of spatial transcriptome is not at the single-cell level. The official Visium chip has four capture areas on a glass slide, each capture area is 6.5mmX6.5mm, and there are 5000 spots in each area. The number of cells will vary according to the type of sample, usually a dozen or dozens of cells.

Q17 What are the main applications of the spatial transcriptome?

A17 Spatial transcriptome studies analyze the spatial location information of tissues, which can help to reveal the signaling pathways and cellular behaviors occurring in the precise spatial range, and resolve the deeper molecular mechanisms in the target biological processes, which are mostly applied in medical-related research, such as oncology, growth and development, pathology, neuroscience, immunology, etc.