Haynes:UPLassay

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Universal Probe Library Assay

Based on the Universal ProbeLibrary Assay Quick Guide from Roche

1. Design your primers

• Each gene you analyze requires a three-part primer set: forward primer, reverse primer, and a UPL probe.
• Use Roche's Assay Design Center to design optimal primers, and to identify the right UPL 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.

2. 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 reference target gene (a housekeeping gene that is 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.
• Hypothetical example: 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. She will use GAPD as the reference gene, using the Roche-supplied primer set. All of the unique reactions she must set up are:
  

REACTION LIST

	Template	Gene 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:	treated cells	ref. gene, GAPD
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	ref. gene, GAPD
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	ref. gene, GAPD

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

PLATE LAYOUT

	1	2	3	4	5	6	7	8	9	10	11	12
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
D	---	---	---	---	---	---	---	---	---	---	---	---
E	---	---	---	---	---	---	---	---	---	---	---	---
F	---	---	---	---	---	---	---	---	---	---	---	---
G	---	---	---	---	---	---	---	---	---	---	---	---
H	---	---	---	---	---	---	---	---	---	---	---	---

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 layout in your notes. Your plate set-up will probably vary for each run.

3. Reaction Set-up: PCR master mixes for each Gene Target

• Create a PCR master mix for every unique primer set.
• In the example above, Gene Target A is included in 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 Gene Targets B, C, and GAPD in separate tubes (each column in the table below represents a 1.5 mL tube).

Reagent	(Single well)	Gene Target A (x10)	Gene Target B (x10)	Gene Target C (x10)	Gene Target GAPD (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 GAPD primers*
20 μM Reverse primer	(0.3 μL)	3.0	3.0	3.0	---
10 μM UPL probe	(0.3 μL)	3.0	3.0	3.0	3.0 GAPD UPL probe*
PCR H2O	(0.1 μL)	1.0	1.0	1.0	4.0
Total vol.	(8.5 μL)	85.0	85.0	85.0	85.0

*GAPD primer mix and the GAPD UPL probe are supplied in the Roche Universal ProbeLibrary Human GAPD Assay kit, #05190541001

4. Reaction Set-up: template cDNA dilutions

• Typically, you will have only 20 μL of stock cDNA on hand. Use a little of the stock cDNA to make a separate dilution of cDNA in order to extend its use. For many reactions, a 1:10 dilution is suitable. For GAPDH, you should use a 1:1000 or 1:10,000 dilution since this gene is expressed at levels so high, it can produce saturating qPCR signals
• In the example above, treated cell cDNA is needed for 4 unique reactions, with 3 technical replicates each. Thus, enough master mix should be made for 4 Rxns x 3 replicates + 1 extra = 13 individual wells (the "extra" is included so that you don't run out of master mix). The same needs to be done for templates "untreated" and "no template" in separate tubes (each column in the table below represents a 1.5 mL tube).

Reagent	(Single well)	treated cDNA (x13)	untreated cDNA (x13)	no template (x13)
diluted cDNA	(2.0 μL)	26.0	26.0	---
PCR H2O	(4.5 μL)	58.5	58.5	84.5
Total vol.	(6.5 μL)	84.5	84.5	84.5

5. Reaction Set-up: loading the 96-well plate
In the end, each well will have a total volume of 15.0 μL. How do we end up with that number?

• In this hypothetical experiment, at this point the scientist has seven 1.5 mL tubes: Gene A, Gene B, Gene C, GAPD, treated cDNA, untreated cDNA, and no template.
• She will pipette 19.5 μL (3 x 6.5) of treated cDNA dilution into well A1.
• She will add 25.5 μL (3 x 8.5) of Gene A PCR master mix to the cDNA in A1, and mix by gently pipetting up and down 3 - 5 times (without making bubbles).
• Well A1 now has 45.0 μL of all of the components for Rxn 1.
• She will use the same pipette tip to transfer 15 μL of solution from A1 into A2, and A3.
• Now wells A1, A2, and A3 each have 15 μL of Rxn 1.
• She will repeat these steps using the appropriate combinations of cDNA template and Primer mix as shown below:

	1	2	3	4	5	6	7	8	9	10	11	12
A	treated/A	"	"	treated/B	"	"	treated/C	"	"	treated/D	"	"
B	untreated/A	"	"	untreated/B	"	"	untreated/C	"	"	untreated/D	"	"
C	NTC/A	"	"	NTC/B	"	"	NTC/C	"	"	NTC/D	"	"
D	---	---	---	---	---	---	---	---	---	---	---	---
E	---	---	---	---	---	---	---	---	---	---	---	---
F	---	---	---	---	---	---	---	---	---	---	---	---
G	---	---	---	---	---	---	---	---	---	---	---	---
H	---	---	---	---	---	---	---	---	---	---	---	---

The plate is now ready to load into the Light Cycler 480!