Haynes:UPLassay

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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
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