I am doing RNA immunoprecipitation, using the RNA TRAP technology, with which you can capture actively translating ribosomes with the L10 ribosomal subunit fused with GFP, using GFP antibody coated magnetic beads. Right now I'm testing it on HEK293T cells transfected with L10-GFP and I am having trouble finding good reference genes / controls for qPCR.

GADPH & b-actin seem to be differentially expressed between the IP & the unbound (the supernatant of the IP) fractions, which result in very different & unreliable results when I look at enrichment of GFP between IP & unbound. For example, b-actin sometimes shows Ct value difference of 5 between IP & unbound fraction, of the sample.. Actually b-actin seems to be 'enriched' in the IP sample (so I suspect it binds to the beads..)

What do you suggest I do? Should I compare with the input sample? n

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