WE have tried PCR (using Lightcycler) but could not detect GCR beta We are now trying to find another technique, looking at flow for example. It is known that beta is around 1000x lower in expression than GCR alpha (the main active form of GCR), so we would need to find a very sensitive means of detecting beta. Any advice would be greatly appreciated. Many thanks.
Hey Fane,
I wouldn't expect too much success for detecting GCR beta protein with conventional flow when you couldn't detect it on RNA level (assuming your primers worked as they were supposed to). If you are in possession of a working antibody against GCR beta, then of course it might still be worth a shot to try and stain for the protein. To amplify the fluorescent signal you could use multistep-labeling using biotin and streptavidin or a secondary polyclonal 'antibody' which binds to multiple sites on your primary antibody in a species-specific manner (e.g. using an anti-rat polyclonal Ab if your primary Ab is from rat etc). Try both surface and intra-cellular staining just to check wether your primary antibody works at all.
If no antibody is available and you still want to go with flow cytometry, then RNA Flow (e.g. QuantiGene from Affymetrix) using a labeled RNA probe (including several steps for signal amplification) could be an alternative.
In case you've tried qPCR using SYBR Green, perhaps try again with a Taqman assay instead, which is to my understanding more specific and in certain instances also more sensitive with respect to the detection limit. As a last resort, you could consider doing RNA-Seq on sorted cells, which of course is somewhat more elaborate.
Hope you'll find a solution!
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