How to explain this:

Go to the profile of samshen
May 22, 2017

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!



Go to the profile of drshabba
drshabba 9 months ago

Are you sure your second signal is from the FAM fluorophore? - 560 is approximately the emission from a HEX labelled probe.

I'm not entirely sure why you are measuring at 560; unless you have a HEX labelled probe for a internal control, or house keeping gene? Either of these is a must if you're trying to compare the relative expression of two genes (in two different reactions).

TAMRA quencher is designed to quench fluorescence produced by the FAM fluorophore when it is excited by the machines light source (and when not released during the reaction).

It would be more useful for you to explain a bit more about your experimental details, when you say "reaction curve" what do you mean?