Spatial OMICS

Question 1

Why would you use Spatial omics?

Answer 1

To find the location of certain cell gene expression. The location might give us useful info. SC analysis will provide alot of transcriptome data on the different cell present in the tumor micro-environment, including differences within 1 cell population. HOWEVER, without the histological info, it is difficult to identify and localize the different subpopulations in respect to the tumor

Question 2

Which 2 methods are the most basic forms of spatial omics

Answer 2

Imaged based and capture based

Question 3

What is the principle of imaged based spatial omics

Answer 3

Imaging-based spatial transcriptomics use in situ hybridization of mRNAs with labelled complementary probes, detected by microscopy to quantify transcripts = smFISH (single molecular fluorescent in situ)

The first step is to fix the cell and permeabilization to help the probes get into the cell. Then hybridize and incubate. The probes will bind to target RNA. Then the cell need to be washed and can be taken under the microscope for imaging (standard fluorescent microscope). Under the microscope you will have to count the dots to see where and how much gene is expressed (might be a limitation)

Question 4

What will help with the limitation of smFISH

Answer 4

cell segmentation or staining the cell border with fluorescence if the cell border is not clear

Question 5

Name the advantage and disadvantage of smFISH

Answer 5

Advantage:
- High coverage for genes
- High resolution (single cell or even subcellular location of transcripts if the tissue is not too complicated)

Disadvantage:
- Rules of light: Diffraction (hard with counting clear single spots/see spots apart)
- Limited detectable colours before colours overlab –> Cyclic labeling may fix this problem (microscopic/probe issue)

Question 6

Explain the cycling labeling method

Answer 6

First, label cell with certain colours, remove the colours (wash) and then label something else with the same colours

Question 7

Explain the capture based method principle (LCM)

Answer 7

LCM= Laser Micro Dissection
A histological section is placed on the stage of the specially designed microscope and the pathologist selects the area of interest. A transparent ethylene vinyl acetate layer is apposed to the section and a low power infrared laser beam is directed at the cells of interest. When the operator activates the laser beam, the film directly above the targeted area transiently melts and the long chain polymers in the film surround and tightly hold the cells, which remain embedded in the film after its removal from the section. After that RNA analysis

Question 8

Explain the capture based method principle (Barcodes Solid Phase RNA capture)

Answer 8

This is the most advanced technique. You don’t use beats, but spots where the location is known. Those spots are in a capture area, filled with primers with barcodes and UMI. Those spots can be compared to beats to catch RNA in the cell. The barcode is used to pinpoint where the RNA came from (capture areas). Then sequencing. All the probes can be the same, but the barcode determines which spot the sequence came from.