As we sometimes see a statistically significant difference does not necessarily mean a difference in actual function. My question is does a receptor mRNA (like 5ht1a) decrease of less than 5% result in decreased functional receptors' level expressed on the membrane surface? How I can prove or disapprove this? How can we be confident over our qRT-PCR data showing the significant change in expression levels?
The answer is "it depends".
Remember, you are generally preparing cDNA from bulk cell preps, so your measurements are necessarily at the population level.
A 5% decrease in an mRNA, in 100% of the cells, likely falls within the range of intracellular stochastic noise, and is probably of no functional consequence.
A 100% decrease in 5% of the cells, however, almost certainly has clear biological consequences for those specific cells.
So, until you can distinguish these two possibilities, it's really impossible to say.
Can you do immunolabelling/FACS for the receptor on cell cultures? If you have marked, specific reductions in sub-populations, maybe you have a story here.
Hi Tabinda,
If you can find good antibody against the extracellular domain of this receptors you could staining the cells without permeabilization so only membrane expose receptor is detected. On top of this, and as John well mentioned above, if you can distinguish those cells undergoing the receptor knockdown (GFP reporter or another type of co-labelling) from the rest of the unmodified population then you can fuether narrow down your search and the specificity of your results.
Cheers,
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