I have many single-cell sequencing data. I checked my gene of interest in these data. However, all of these single-cell sequencing data showed that my gene has no expression, no matter in immune population or tumor population.
The problem is, I can detect its expression in my cancer cell line and some immune cells both in RNA level and in protein level.
But why single-cell data showed it has no expression at all??
Hi,
A few things that might explain it>
1. If the half life of the RNA is short, the procedure to isolate the single cells may be causing expression to turn off and its degraded before the RNA is isolated.
2. It may be expressed (perhaps at a high level) in a small percentage of the cells, and you have not captured one of these in your single cell experiments.
3. It amplifies poorly compared to other transcripts. The number of rounds of amplification during library preparation for single cell methods can be high compared to other methods, a poorly amplifying gene may drop out.
If you have a good antibody you could use either a flow or immunohistochemistry experiment to see if the percentage of cells in the population expressing the protein is small (you could also do this by generating probes for RNA FISH).
Good Luck!
Frank
Hi Suipwksiow,
in addition to the good analysis from Frank R Burns, you could simply check the level of expression of your target (see GEO profile or genecards) or see if your gene region is GC rich (in this case, capture would give you better results than amplification during the library preparation).
fred
Maybe that single cell happens to be non-cancerous? Where does it come from?
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