Luminescence: Background or real signal? - (Apr/26/2013 )

Dear all,

I am confused about some sensitivity/gain - background issues.

We use a dual luciferase based assay system to measure gene expression. Now some of the genes are expressed very high, some are expressed very low. The latter show so low values it seems to be below background values, no matter what blank measurements I make (air, water, buffer+substrate).

From the literature I know the principle how photomultiplier tubes (PMT) should work. Therefore, a signal increase seen by increasing the gain is an artefact, i.e. increasing gain will not increase the input.

However, when I compare the measured signals with our expectations/hypothesis, meaning the regulation pattern of the genes, it is as expected. We repeated these experiments at least 10 - 20 times and it is highly significant by t-test statistics (as well as by looking at the data by eye). So the results fit our model, but in theory they shouldn't... Correct?

How can this be explained? Has anybody a good idea, knows some good literature? Are we producing simply artifacts by chance?

Thank you for any help to make me understanding,

cheers

hmm.... as far as your low values, if they are below your "blank" or negative control values, you need a different negative control. can you list in detail what you have tried?

i tried as a negative control to measure wells with buffer+substrate w/o any cell lysate, the same with untreated cell lysate. also I measured empty wells (air), I measured wells containing water, I measured wells with lysis buffer only. so there I don't see any further possibilities for blanks that make any sense or would improve my results. do you have any suggestions? or can you help me to understand the discrepancy between theory (no possible signal increase) and practice (correct/expected observations)?
thanks for your time