Haynes:UPLassay

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 A single plate contains 96 wells, as shown below   1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H Reaction Set-up Create a PCR master mix for every unique primer mix