We need a model to transfect TLR4 and all sources say that HEK293 is the one, but when we are looking to confirm it, we detect TLR4 mRNA and we do not find the bibliographic source that says unequivocally that HEK293 does not express TLR4 at any level. Where are we getting it wrong?
How much are you detecting? How many cycles for the Rt-PCR? Make sure you have tried more than one primer pair, that your melting curves are ideal, and that you are getting the right size PCR product. Also you can inlcude a standard template to determine copy number. A typical gene at medium levels may have 1000-10,000 copies per cell. If you have less than 100, then you are at the low end of expression. You may not find a definitive paper on this, so trust your own data.
I agree with Gregory. Trust your own data. You are probably getting it right. It is likely that HEK293 express some level of TLR4 (it may be very difficult to find a cell line that expresses absolutely no TLR4). Sources may indicate no TLR4 expression but if so then the expression is likely relative to a cell line that expresses high amounts of TLR4 mRNA or protein. Moreover, their assays may not be sensitive enough to detect the TLR4 (Western blot not exposed long enough, etc). Sources may also be basing their results on experiments indicating very low or undetectable response to LPS in untransfected HEK293...probably using an NF-kB reporter construct to test the LPS response. Yet, this doesn't necessarily mean that HEK293 express no TLR4.
I agree with both Gregory and Erik, trust your data. The reason why paper are scanty on such may be because of insignificant level of detection. Best wishes
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