SpaRTAN

Wax tissue blocks

• Ideally, select archive wax tissue blocks that have been stored at 4°C to ensure to preserve RNA integrity. Please do not select blocks where the tissue is extremely thin.
• It is a good idea to look at any original H&E images before sending to verify that tissue morphology is good.
• Ensure that the area of interest will fit within the Capture Area of a Visium CytAssist Spatial Gene Expression Slide. Tissue sections larger than these Capture Areas may be placed on the slide, but only the tissue within the Capture Area will be processed by the CytAssist instrument.

• HDBR will assess RNA quality of the tissue block or archived sections before proceeding with sectioning by calculating the percentage of total RNA fragments >200 nucle­otides (DV200) of RNA extracted from tissue sections.

Unstained Wax embedded tissue sections on glass slides

• Over time, archived slides may experience RNA degradation; therefore, freshly placed FFPE tissue sections are preferred for the Visium CytAssist assay.
• Unstained glass slides containing tissue of interest can be received by HDBR. Ideally, select slides that have been stored at 4°C to ensure to preserve RNA integrity. 
• Sections must be placed on blank slides that are centred within the appropriate Capture Area aligned to the Tissue Slide Stage on the Visium CytAssist instrument (see diagram below)

• HDBR will assess RNA quality of the archived sections via scraping off tissue sections before proceeding with the CytAssist protocol by calculating the percentage of total RNA fragments >200 nucle­otides (DV200) of RNA extracted from tissue sections.

Hardset H&E Archived Slides

• Over time, archived slides may experience RNA degradation; therefore, freshly placed FFPE tissue sections are preferred for the Visium CytAssist assay.
• Stained glass slides containing tissue of interest can be received by HDBR
• Sections must be placed on blank slides that are centered within the appropriate Capture Area aligned to the Tissue Slide Stage on the Visium CytAssist instrument. Diagrams for verifying that tissue sections are placed in the allowable area can also be found in the Visium CytAssist Quick Reference Cards - Acces­sory Kit (Document CG000548). 
  • HDBR will assess RNA quality of the archived sections via scraping off tissue sections before proceeding with the CytAssist protocol by calculating the percentage of total RNA fragments >200 nucleotides (DV200) of RNA extracted from tissue sections.

Provided the tissue passes QC checks you should have results in the form of an electronic report within 6-8 weeks of the project starting. The exact time a project commences will depend upon the workload of the team at that time and the scale of the study. The sooner the tissue is received, the sooner it can join the queue of projects.

For frozen tissue, and standard definition, 4 sections will fit into four 6.5 x 6.5mm capture areas on the slide.

For FFPE tissue, sections already on microscope slide, or Visium HD, 2 sections will fit into either two 6.5 x 6.5mm or two 11 x 11cm capture areas on the slide.

Yes, a new modification of the technique using a CytAssist instrument can help with this however the tissue placement and quality of the tissue would have to be checked first.

or Standrad Visium (version 1) the capture area is 6.5 x 6.5mm. There are a total of 4992 total spots per capture area and each spot is 55µm in diameter with a 100µm centre-to-centre distance between spots.

Each spot contains in the order of millions of probes.

The number of cells captured in a single spot is based upon the tissue type, cell size and section thickness, this is generally between 1-10 cells.

For Visium HD, the resolution is increased to 2µm resolution, with no gaps on the slide.

We would ideally require a RNA DV200 value of at least 30% to be able to proceed.

The Visium Spatial Gene Expression solution is designed to enable unbiased whole transcriptome analysis of FFPE tissue sections. In fresh frozen tissue, the RNA is permeabilized so that it can bind the spots. In contrast, in the Visium for FFPE assay, an extra step is necessary to capture the gene expression information. In Visium for FFPE, probe pairs target specific sequences of the RNA. The probe pairs target around 18,000 genes in both human and mouse samples.

The sensitivity of the Visium Spatial Gene Expression solution is dependent on a large number of factors, such as tissue type, species, permeabilization time, and sequencing depth. It is challenging to predict the sensitivity of a given experiment beforehand, but a successful Tissue Optimization experiment with a good fluorescent signature will help guide for optimal gene expression data and experimental outcome.

For example, if the percentage of capture area covered by the tissue section is 60% and there are 5,000 total spots on the capture area, the total sequencing depth required would be:

(0.60 x 5,000 total spots) x 50,000 read pairs/spot = 150 million total read pairs for that sample

he Visium Spatial Gene Expression solution produces spatially barcoded, Illumina sequencer-ready libraries. The minimum recommended sequencing depth for Visium Spatial Gene Expression libraries is 50k read pairs per spot covered with tissue for fresh frozen tissues, 25k per spot for FFPE tissues and CytAssist analysis, and  275M reads per window for VisiumHD.