I'm working with C6 glial cell line and challenging with LPS, ATP and BZATP. But in the control, getting the expression of COX-2 band. What could be the reason??
Are you doing quantitative PCR or endpoint PCR? or are you doing a western-blot? You just mention "a band"
If you are doing real-time quantitative PCR, what are the fold differences in copies corrected for reference gene expression, amplification efficiency etc. If you are doing endpoint PCR, how many cycles are you using? Have you determined a cut-off point for the sensitivity of your assay? Nearly all genes are expressed at some basal level, so you will expect some amplification if you do enough rounds of PCR.
LPS should induce Cox-2 at a higher level than control if TLR4 /MD2 is expressed in these lines. For the sake of argument if your LPS PCR amplification reaches a plateau after 20 cycles, and there are 1000x more Cox-2 molecules than your control - it will plateau at about 30 cycles, then doing 35-40 cycles of PCR and resolving the products on a gel will give the impression that levels are similar in both, because they have both plateaued. You need to determine at which cycles both your control and treated cells produce detectable product.
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Have you done a time course of expression? eg 3h 6h etc etc? This will make a difference.as to the differences you see.
Ian
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