I want to make some single-cell RT-qPCR or scRNA-Seq experiments. But, I'm affraid, that during tissue dissociatation and FACS sorting are cells stressed, and it's activate expression of some genes, which we want to study.
So, in my idea, I want to first make tissue fixation, after that dissociate cells and continue with sorting/analysis. Have anybody experience with protocol like this?
Have you thought about using Laser Microdissection? You would need to prepare tissue sections (using a microtome or cryotome) and then fix the tissue on the slide. At the microscope, you select the cells of interest for precise dissection. The cells are excised and then collected under full visual control.
The great advantage is that the cells are not stressed by any solvents for solubilization. More importantly, you will know the spatial origin of your isolated cells.
First, the most important disadvantage of Laser Microdissection is quality of RNA from these cells. Second problem, it's not compatible with droplet based method for single-cell RNA-Seq library prep.
I agree with your second issue. For droplet based methods you require cells in suspension.
I also have to admit that I heard rumors that RNA quality is low for microdissected samples. However, MMI is using a low-power laser to avoid any DNA/RNA damages. Importantly, there are plenty of publications using laser microdissected samples for RNA analysis (spatial transcriptomics, RNA seq, ...)
Article Optimized RNA Extraction from Non-deparaffinized, Laser-Micr...
Article Recommendations for mRNA analysis of micro-dissected glomeru...
Article Spatial transcriptomic analysis of cryosectioned tissue samp...
Article Combining laser microdissection and RNA-seq to chart the tra...
Chapter Adaptation of Laser Microdissection Technique to Nanostring ...
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