Universal Probe Library Assay
Based on the Universal Probe Library Assay Quick Guide from Roche
Design your primers
- Each gene you analyze requires a primer set: 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 reactions
How many reactions should I plan to run? Each experimental cDNA sample is a template. The gene being detected is often referred to as a target. You should also include a loading control target such as the GAPDH or actin housekeeping genes (always active, not expected to change). 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:
|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:||treated cells||loading control, primer set D|
|Rxn 5:||untreated cells||gene A, primer set A|
|Rxn 6:||untreated cells||gene B, primer set B|
|Rxn 7:||untreated cells||gene C, primer set C|
|Rxn 8:||untreated cells||loading control, primer set D|
|Rxn 9:||no template||gene A, primer set A|
|Rxn 10:||no template||gene B, primer set B|
|Rxn 11:||no template||gene C, primer set C|
|Rxn 12:||no template||loading control, primer set D|
This hypothetical experiment requires 12 total unique reactions.
A single plate contains 96 wells (as shown below). To insure accuracy, three technical replicates per reaction (Rxn) are required
|A||Rxn 1||Rxn 1||Rxn 1||Rxn 2||Rxn 2||Rxn 2||Rxn 3||Rxn 3||Rxn 3||Rxn 4||Rxn 4||Rxn 4|
|B||Rxn 5||Rxn 5||Rxn 5||Rxn 6||Rxn 6||Rxn 6||Rxn 7||Rxn 7||Rxn 7||Rxn 8||Rxn 8||Rxn 8|
|C||Rxn 9||Rxn 9||Rxn 9||Rxn 10||Rxn 10||Rxn 10||Rxn 11||Rxn 11||Rxn 11||Rxn 12||Rxn 12||Rxn 12|
This hypothetical experiment requires 12 Rxns x 3 replicates = 36 wells. If you need more than 96 wells, you must split the experiment over multiple plates. This plate is set up so that there is one template per row, and a target for every three columns. You can use whatever organization suits your experiment. It is absolutely critical that you keep a reaction list and plate table in your notes. Your plate et-up will probably vary for each run.
Reaction Set-up: PCR master mixes for each gene target
- Create a PCR master mix for every unique primer set.
- In the example above, primer set A is needed for 3 unique reactions, with 3 technical replicates each. Thus, enough master mix should be made for 3 Rxns x 3 replicates + 1 extra = 10 individual wells (the "extra" is included so that you don't run out of master mix). The same needs to be done for primer sets B, C, and D in separate tubes (each column in the table below is a 1.5 mL tube).
|Reagent||Single well||Gene A (x10)||Gene B (x10)||Gene C (x10)||Loading ctrl gene D (x10)|
|2x LC480 Probes Master||7.5 μL||75.0||75.0||75.0||75.0|
|20 μM Forward primer||0.3 μL||3.0||3.0||3.0||3.0|
|20 μM Reverse primer||0.3 μL||3.0||3.0||3.0||3.0|
|10 μM UPL probe||0.3 μL||3.0||3.0||3.0||3.0|
|PCR H2O||5.1 μL||51.0||51.0||51.0||51.0|
|Total vol.||8.5 μL||85.0||85.0||85.0|| 85.0