How to explain this:
We have a Q-PCR test that compare a gene with another. Both probes are labeled with same pair of report fluorescent and quencher (FAM/TAMRA). Reaction of two genes are separated (not multi-color in one reaction tube). Tests are run by Roche light cycler 2.0. One gene (gene 1) has very nice reaction curve at both channel 1 (530) , which is expected, and a nice curve channel 2 (560), which almost run parallel to channel one. The other (gene 2) has only about 10% light density at channel 1 (530), but is nice at channel 2 (560), which is similar to the gene 1. Here are some questions:
1. For gene 1, Nice reaction curve at channel 1 (530) is expected, however, how to explain the nice reaction curve at channel 2 (560)?
2. For gene 2, why reaction curve at channel 1 is very weak, while reaction curve at channel 2 is good?
3. Roche light cycler 2.0 only has one blue LED as a excitation energy source, what excite TAMRA to produce light at 560 nm?
4. Why TAMRA also show a reaction curve almost parallel to FAM curve in both genes' reactions, but one is also has similar light density, while other channel 1 only as about 10% strong as channel 2?
Thanks for any comments!