Haynes:UPLassay
From OpenWetWare
Universal Probe Library Assay
Based on the Universal Probe Library Assay Quick Guide from Roche
Design your primers
- Each gene you analyze requires a forward primer, reverse primer, and a UPL probe.
- Use Roche's Assay Design Center to design optimal primers and identify the right probe for your gene(s) of interest.
- The forward and reverse primers need to be ordered from a DNA synthesis company (e.g., IDT DNA, Promega, etc.), and the UPL oligo comes from Roche.
Design your experiment
How many reactions should I plan to run? Each experimental sample is a template. The gene being detected is often referred to as a target. Each unique template and target combination requires its own reaction. You will also need to set up a no template control to observe the amount of background noise from that reaction. For instance, a scientist wants to measure differences the expression of genes A, B, and C in an experiment where cells were treated with a drug, or untreated. All of the unique reactions she must set up are:
Template | Target | ||||||||||||||||||||||
Rxn 1: | treated cells | gene A, primer set A | |||||||||||||||||||||
Rxn 2: | treated cells | gene B, primer set B | |||||||||||||||||||||
Rxn 3: | treated cells | gene C, primer set C | |||||||||||||||||||||
Rxn 4: | untreated cells | gene A, primer set A | |||||||||||||||||||||
Rxn 5: | untreated cells | gene B, primer set B | |||||||||||||||||||||
Rxn 6: | untreated cells | gene C, primer set C | |||||||||||||||||||||
Rxn 7: | no template | gene A, primer set A | |||||||||||||||||||||
Rxn 8: | no template | gene B, primer set B | |||||||||||||||||||||
Rxn 9: | no template | gene C, primer set C
|