Haynes:UPLassay

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(New page: '''Universal Probe Library Assay'''<br> Based on the Universal Probe Library Assay Quick Guide from Roche '''Design your primers'''<br> * Each gene you analyze requires a '''forward prim...)
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* Use Roche's [http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000 Assay Design Center] to design optimal primers and identify the right probe for your gene(s) of interest.
* Use Roche's [http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000 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.
* 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.
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'''Design your experiment'''<br>
'''Design your experiment'''<br>
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''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:<br>
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''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'''. 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:<br>
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{|
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{| class="wikitable" style="width: 300px; height: 200px;"
|-
|-
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| &nbsp; || Template || Target  
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| &nbsp; || '''Template''' || '''Target'''
|-
|-
|Rxn 1: || treated cells || gene A, primer set A
|Rxn 1: || treated cells || gene A, primer set A
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| Rxn 3: || treated cells || gene C, primer set C
| Rxn 3: || treated cells || gene C, primer set C
|-
|-
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| Rxn 4: || untreated cells || gene A, primer set A
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| Rxn 4: || treated cells || loading control, primer set D
|-
|-
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| Rxn 5: || untreated cells || gene B, primer set B
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| Rxn 5: || untreated cells || gene A, primer set A
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|-
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| Rxn 6: || untreated cells || gene B, primer set B
|-  
|-  
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| Rxn 6: || untreated cells || gene C, primer set C
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| Rxn 7: || untreated cells || gene C, primer set C
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|-
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| Rxn 8: || untreated cells || loading control, primer set D
|-  
|-  
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| Rxn 7: || no template || gene A, primer set A
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| Rxn 9: || no template || gene A, primer set A
|-
|-
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| Rxn 8: || no template || gene B, primer set B
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| Rxn 10: || no template || gene B, primer set B
|-
|-
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| Rxn 9: || no template || gene C, primer set C
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| Rxn 11: || no template || gene C, primer set C
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|-
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| Rxn 12: || no template || loading control, primer set D
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|}
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This hypothetical experiment requires 12 total unique reactions.<br>
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A single plate contains 96 wells, as shown below
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A single plate contains 96 wells (as shown below). To insure accuracy, '''three technical replicates per reaction''' (Rxn) are required
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{| class="wikitable" style="width: 500px; height: 200px;"
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{| class="wikitable" style="width: 600px; height: 200px;"
|-
|-
| &nbsp; || 1 || 2 || 3 || 4 || 5 || 6 || 7 || 8 || 9 || 10 || 11 || 12
| &nbsp; || 1 || 2 || 3 || 4 || 5 || 6 || 7 || 8 || 9 || 10 || 11 || 12
|-
|-
| A
| A
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| style="background: lightgrey" | Rxn 1
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| style="background: lightgrey" | Rxn 1
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| style="background: lightgrey" | Rxn 1
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| style="background: pink" | Rxn 2
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| style="background: pink" | Rxn 2
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| style="background: pink" | Rxn 2
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| style="background: lightgreen" | Rxn 3
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| style="background: lightgreen" | Rxn 3
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| style="background: lightgreen" | Rxn 3
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| style="background: lightblue" | Rxn 4
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| style="background: lightblue" | Rxn 4
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| style="background: lightblue" | Rxn 4
|-
|-
| B
| B
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'''Reaction Set-up'''
'''Reaction Set-up'''
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* Create a PCR master mix for every unique primer mix
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* Create a PCR master mix for every unique primer mix. In the example above, primer set A is needed for

Revision as of 20:15, 17 May 2012

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. 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:

  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: 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

  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
C
D
E
F
G
H


Reaction Set-up

  • Create a PCR master mix for every unique primer mix. In the example above, primer set A is needed for
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