Haynes:UPLassay

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'''Universal Probe Library Assay'''<br>
+
<- [[Haynes:Protocols | Back to Protocols]]
-
Based on the Universal Probe Library Assay Quick Guide from Roche
+
 +
=Universal Probe Library Assay=
 +
Based on the Universal ProbeLibrary Assay Quick Guide from Roche
-
'''Design your primers'''<br>
+
<div style="width:800px;">
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* Each gene you analyze requires a primer set: '''forward primer''', '''reverse primer''', and a '''UPL probe'''.  
+
 
-
* 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.
+
Workflow Overview
 +
# Planning: Design your primers
 +
# Planning: Design your reactions
 +
# Wet bench: Make Gene Target master mixes
 +
# Wet bench: Make Template (cDNA) master mixes separately
 +
# Wet bench: Add the appropriate combinations of of the master mixes to the 96-well plate
 +
---> Run the reaction in the Light Cycler!
 +
 
 +
 
 +
----
 +
 
 +
1. '''Design your primers'''<br>
 +
* Each gene you analyze requires a set of three oligos: '''forward primer''', '''reverse primer''', and a '''UPL probe'''.  
 +
* Use Roche's [http://www.roche-applied-science.com/webapp/wcs/stores/servlet/CategoryDisplay?catalogId=10001&tab=Assay+Design+Center&identifier=Universal+Probe+Library&langId=-1#tab-3 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.
* 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'''<br>
+
----
-
''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:<br>
+
-
{| class="wikitable" style="width: 400px; height: 200px; border: 1px"
+
2. '''Set up a reaction list and plan the plate layout'''<br>
 +
* ''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 '''Gene Target'''. You should also include a '''reference''' Gene Target such as GAPD (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:
 +
** cDNA samples + a no template control = 3
 +
** Gene targets + GAPD reference = 5
 +
** Replicates per reaction = 3
 +
** '''Wells needed = 3 * 5 * 3 = 45'''
 +
 
 +
 
 +
{| class="wikitable" style="width: 800px;"
 +
|- valign="top"
 +
| '''REACTION LIST'''
 +
{| width=300px
|-
|-
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| &nbsp; || '''Template''' || '''Target'''
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| &nbsp; || '''Template cDNA''' || '''Gene Target'''
|-
|-
-
|Rxn 1: || treated cells || gene A, primer set A
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| Rxn 1: || treated cells || MPK14
|-
|-
-
| Rxn 2: || treated cells || gene B, primer set B
+
| Rxn 2: || treated cells || CBX8
|-
|-
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| Rxn 3: || treated cells || gene C, primer set C
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| Rxn 3: || treated cells || TNF
|-
|-
-
| Rxn 4: || treated cells || loading control, primer set D
+
| Rxn 4: || treated cells || NPPA
|-
|-
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| Rxn 5: || untreated cells || gene A, primer set A
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| Rxn 5: || treated cells || GAPD (reference gene)
|-
|-
-
| Rxn 6: || untreated cells || gene B, primer set B
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| Rxn 6: || untreated cells || MPK14
-
|-
+
-
| Rxn 7: || untreated cells || gene C, primer set C
+
|-
|-
-
| Rxn 8: || untreated cells || loading control, primer set D
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| Rxn 7: || untreated cells || CBX8
-
|-
+
-
| Rxn 9: || no template || gene A, primer set A
+
|-
|-
-
| Rxn 10: || no template || gene B, primer set B
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| Rxn 8: || untreated cells || TNF
|-
|-
-
| Rxn 11: || no template || gene C, primer set C
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| Rxn 9: || untreated cells || NPPA
|-
|-
-
| Rxn 12: || no template || loading control, primer set D
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| Rxn 10: || untreated cells || GAPD (reference gene)
-
|}
+
-
 
+
-
This hypothetical experiment requires 12 total unique reactions.<br>
+
-
 
+
-
 
+
-
A single plate contains 96 wells (as shown below). To insure accuracy, '''three technical replicates per reaction''' (Rxn) are required
+
-
 
+
-
{| class="wikitable" style="width: 600px; height: 200px;"
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|-
|-
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| &nbsp; || 1 || 2 || 3 || 4 || 5 || 6 || 7 || 8 || 9 || 10 || 11 || 12
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| Rxn 11: || no template || MPK14
|-
|-
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| A
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| Rxn 12: || no template || CBX8
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| style="background: silver" | Rxn 1
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-
| style="background: silver" | Rxn 1
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-
| style="background: silver" | 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
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|-
|-
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| B
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| Rxn 13: || no template || TNF
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| style="background: yellow" | Rxn 5
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-
| style="background: yellow" | Rxn 5
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-
| style="background: yellow" | Rxn 5
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-
| style="background: lavender" | Rxn 6
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-
| style="background: lavender" | Rxn 6
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-
| style="background: lavender" | Rxn 6
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-
| style="background: orange" | Rxn 7
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-
| style="background: orange" | Rxn 7
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-
| style="background: orange" | Rxn 7
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-
| style="background: aqua" | Rxn 8
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-
| style="background: aqua" | Rxn 8
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-
| style="background: aqua" | Rxn 8
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|-
|-
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| C
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| Rxn 14: || no template || NPPA
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| style="background: orchid" | Rxn 9
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-
| style="background: orchid" | Rxn 9
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-
| style="background: orchid" | Rxn 9
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-
| style="background: tan" | Rxn 10
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-
| style="background: tan" | Rxn 10
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-
| style="background: tan" | Rxn 10
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-
| style="background: lime" | Rxn 11
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-
| style="background: lime" | Rxn 11
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-
| style="background: lime" | Rxn 11
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-
| style="background: ivory" | Rxn 12
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-
| style="background: ivory" | Rxn 12
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-
| style="background: ivory" | Rxn 12
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|-
|-
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| D
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| Rxn 15: || no template || GAPD (reference gene)
-
|-
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|}
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| E
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| '''PLATE LAYOUT'''<br>
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|-
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Variation 1<br> [[Image:Haynes_UPL_fig1.png|300px|Figure 1]] <br>
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| F
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Variation 2<br> [[Image:Haynes_UPL_fig2.png|300px|Figure 1]]<br>
-
|-
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Both Variations (1 and 2) are correct. Choose a format that helps you to easily keep track of the samples. If you have a large experiment, try to fit as many reactions on the plate as you can (to avoid wasting plates), but also keep the samples arranged in an orderly fashion so that the set-up won't confuse you.
-
| G
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-
|-
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-
| H
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|}
|}
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This hypothetical experiment requires '''12 Rxns x 3 replicates = <u>36 wells</u>'''. 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.
+
* A single plate contains 96 wells. To insure accuracy, '''three technical replicates per reaction''' (Rxn) are required
 +
* If you need more than 96 wells, you must split the experiment over multiple plates.  
 +
* 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.
-
'''Reaction Set-up: PCR master mixes for each gene target'''<br>
+
----
-
* 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 = <u>10 individual wells</u>''' (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).
+
3. '''Reaction set-up: PCR master mixes for each Gene Target'''<br>
 +
* Label one 1.5 mL tube per gene target
 +
* Make enough PCR master mix for your plate...  
 +
** '''MPK14''' is in Reactions 1, 6, and 11 = 3
 +
** Replicates per reaction = 3
 +
** '''Master mix amount = 3 * 3 + 1 (to allow for pipetting error) = 10'''  
 +
** The same needs to be done for CBX8, TNF, NPPA, and GAPD in separate tubes.
 +
 
{| class="wikitable"
{| class="wikitable"
-
| <u>Reagent</u> || <u>Single well</u> || <u>Gene A (x10)</u> || <u>Gene B (x10)</u> || <u>Gene C (x10)</u> || <u>Loading ctrl gene D (x10)</u>
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| <u>Reagent</u> || <u>(Single well)</u> || <u>Gene Target (x10)</u> || <u>GAPD (x10)</u>
|-
|-
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| 2x LC480 Probes Master || 7.5 μL || 75.0 || 75.0 || 75.0 || 75.0  
+
| 2x LC480 Probes Master || (7.5 μL) || 75.0 || 75.0  
|-
|-
-
| 20 μM Forward primer || 0.3 μL || 3.0 || 3.0 || 3.0 || 3.0
+
| 20 μM Forward primer || (0.3 μL) || 3.0 || 3.0 GAPD primers*
|-
|-
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| 20 μM Reverse primer || 0.3 μL || 3.0 || 3.0 || 3.0 || 3.0
+
| 20 μM Reverse primer || (0.3 μL) || 3.0 || ---
|-
|-
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| 10 μM UPL probe || 0.3 μL || 3.0 || 3.0 || 3.0 || 3.0
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| 10 μM UPL probe || (0.3 μL) || 3.0 || 3.0 GAPD UPL probe*
|-
|-
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| PCR H<sub>2</sub>O || 5.1 μL || 51.0 || 51.0 || 51.0 || 51.0
+
| PCR H<sub>2</sub>O || (0.1 μL) || 1.0 || '''4.0'''
|-
|-
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| Total vol. || '''8.5 μL''' || '''85.0''' || '''85.0''' || '''85.0''' || '''85.0'''
+
| Total vol. || ('''8.5 μL''') || '''85.0''' || '''85.0'''
|}
|}
 +
''*GAPD primer mix and the GAPD UPL probe are supplied in the Roche Universal ProbeLibrary Human GAPD Assay kit, #05190541001''
-
'''Reaction Set-up: template cDNA dilutions'''<br>
+
Resulting 1.5 mL tubes:
-
* Typically, you will have only 20 μL of stock cDNA on hand. You use a little of the stock cDNA to make a separate dilution of cDNA 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
+
* '''MPK14''' - 85.0 μL
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* 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 = <u>13 individual wells</u>''' (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 is a 1.5 mL tube).
+
* '''CBX8''' - 85.0 μL
 +
* '''TNF''' - 85.0  μL
 +
* '''NPPA''' - 85.0  μL
 +
* '''GAPD''' - 85.0  μL
 +
 
 +
----
 +
 
 +
4. '''Reaction set-up: master mixes for each Template'''<br>
 +
* Typically, you will have only 20 μL of stock cDNA on hand.  
 +
* Make a 1:10 dilution of cDNA by adding 10 μL of the stock cDNA to 90 μL of PCR H<sub>2</sub>O.
 +
* Make enough Template master mix for your plate...
 +
** '''Treated cells cDNA''' is in Reactions 1, 2, 3, 4 and 5 = 5
 +
** Replicates per reaction = 3
 +
** '''Master mix amount = 5 * 3 + 1 (to allow for pipetting error) = 16'''  
 +
** The same needs to be done for templates "untreated cells" and "no template" in separate tubes.
{| class="wikitable"
{| class="wikitable"
-
| <u>Reagent</u> || <u>Single well</u> || <u>treated cDNA (x13)</u> || <u>untreated cDNA (x13)</u> || <u>no template (x13)</u>
+
| <u>Reagent</u> || <u>(Single well)</u> || <u>cDNA Template (x16)</u>
|-
|-
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| diluted cDNA || 2.0 μL || 26.0 || 26.0 || ---
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| 1:10 cDNA dilution || (2.0 μL) || 32.0*
|-
|-
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| PCR H<sub>2</sub>O || 4.5 μL || 58.5 || 58.5 || 84.5
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| PCR H<sub>2</sub>O || (4.5 μL) || 72.0
|-
|-
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| Total vol. || '''6.5 μL''' || '''84.5''' || '''84.5''' || '''84.5'''
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| Total vol. || ('''6.5 μL''') || '''104.0'''
|}
|}
 +
''*For the no template control, use PCR H<sub>2</sub>O instead of cDNA.''
 +
Resulting 1.5 mL tubes:
 +
* '''T1''' - treated cells cDNA, 84.5 μL
 +
* '''T2''' - untreated cells cDNA, 84.5 μL
 +
* '''T3''' - no template control, 84.5  μL
-
'''Reaction Set-up: loading the 96-well plate'''<br>
 
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Each well will have a total volume of 15.0 μL. How do we end up with that number?
 
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* In this hypothetical experiment, at this point the scientist has seven 1.5 mL tubes: Gene A, Gene B, Gene C, Loading ctrl gene D, treated cDNA, untreated cDNA, and no template.
 
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* She will pipette 19.5 μL (3 x 6.5) of '''treated cDNA''' dilution to the PCR mix in A1.
 
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* She will add 25.5 μL (3 x 8.5) of '''Gene A''' PCR master mix into well 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.
 
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* She will repeat these steps using the appropriate combinations of cDNA template and Primer mix as shown below:
 
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{| class="wikitable" style="width: 400px; height: 200px; border: 1px"
+
----
-
|-
+
 
-
| &nbsp; || 1 || 2 || 3 || 4 || 5 || 6 || 7 || 8 || 9 || 10 || 11 || 12
+
5. '''Reaction set-up: loading the 96-well plate'''<br>
-
|-
+
The following illustrations use Plate Layout Variation 2...
-
| A
+
 
-
| style="background: silver" | treated/A
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{| width=800px
-
| style="background: silver" | "
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|- valign="top"
-
| style="background: silver" | "
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| Pipette 19.5 μL of cDNA Template master mixes into the first well of each 3-well group<br>
-
| style="background: pink" | treated/B
+
| align="right" | [[Image:Haynes_UPL_fig3.png|300px|Figure 3]]
-
| style="background: pink" | "
+
|- valign="top"
-
| style="background: pink" | "
+
| Add 25.5 μL of Gene Target master mix to the cDNA. After each addition, mix by gently pipetting up and down 3 - 5 times (without making bubbles).
-
| style="background: lightgreen" | Rxn 3
+
| align="right" | [[Image:Haynes_UPL_fig4.png|350px|Figure 4]]
-
| style="background: lightgreen" | Rxn 3
+
|- valign="top"
-
| style="background: lightgreen" | Rxn 3
+
| Transfer 15 μL of solution from A1 into A2, and A3. Use a fresh pipette tip to do the same for A4-6, and A7-8.
-
| style="background: lightblue" | Rxn 4
+
| align="right" | [[Image:Haynes_UPL_fig5.png|300px|Figure 5]]
-
| style="background: lightblue" | Rxn 4
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|- valign="top"
-
| style="background: lightblue" | Rxn 4
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| Repeat this procedure for the rest of the plate
-
|-
+
| align="right" | [[Image:Haynes_UPL_fig2.png|350px|Figure 2]]
-
| B
+
-
| style="background: yellow" | Rxn 5
+
-
| style="background: yellow" | Rxn 5
+
-
| style="background: yellow" | Rxn 5
+
-
| style="background: lavender" | Rxn 6
+
-
| style="background: lavender" | Rxn 6
+
-
| style="background: lavender" | Rxn 6
+
-
| style="background: orange" | Rxn 7
+
-
| style="background: orange" | Rxn 7
+
-
| style="background: orange" | Rxn 7
+
-
| style="background: aqua" | Rxn 8
+
-
| style="background: aqua" | Rxn 8
+
-
| style="background: aqua" | Rxn 8
+
-
|-
+
-
| C
+
-
| style="background: orchid" | Rxn 9
+
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| style="background: orchid" | Rxn 9
+
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| style="background: orchid" | Rxn 9
+
-
| style="background: tan" | Rxn 10
+
-
| style="background: tan" | Rxn 10
+
-
| style="background: tan" | Rxn 10
+
-
| style="background: lime" | Rxn 11
+
-
| style="background: lime" | Rxn 11
+
-
| style="background: lime" | Rxn 11
+
-
| style="background: ivory" | Rxn 12
+
-
| style="background: ivory" | Rxn 12
+
-
| style="background: ivory" | Rxn 12
+
-
|-
+
-
| D
+
-
|-
+
-
| E
+
-
|-
+
-
| F
+
-
|-
+
-
| G
+
-
|-
+
-
| H
+
|}
|}
 +
 +
* Seal the plate with clear film.
 +
* The plate is now ready to load into the Light Cycler 480!

Current revision

<- Back to Protocols

Universal Probe Library Assay

Based on the Universal ProbeLibrary Assay Quick Guide from Roche

Workflow Overview

  1. Planning: Design your primers
  2. Planning: Design your reactions
  3. Wet bench: Make Gene Target master mixes
  4. Wet bench: Make Template (cDNA) master mixes separately
  5. Wet bench: Add the appropriate combinations of of the master mixes to the 96-well plate

---> Run the reaction in the Light Cycler!



1. Design your primers

  • Each gene you analyze requires a set of three oligos: 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. Set up a reaction list and plan the plate layout

  • 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 Gene Target. You should also include a reference Gene Target such as GAPD (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:
    • cDNA samples + a no template control = 3
    • Gene targets + GAPD reference = 5
    • Replicates per reaction = 3
    • Wells needed = 3 * 5 * 3 = 45


REACTION LIST
  Template cDNA Gene Target
Rxn 1: treated cells MPK14
Rxn 2: treated cells CBX8
Rxn 3: treated cells TNF
Rxn 4: treated cells NPPA
Rxn 5: treated cells GAPD (reference gene)
Rxn 6: untreated cells MPK14
Rxn 7: untreated cells CBX8
Rxn 8: untreated cells TNF
Rxn 9: untreated cells NPPA
Rxn 10: untreated cells GAPD (reference gene)
Rxn 11: no template MPK14
Rxn 12: no template CBX8
Rxn 13: no template TNF
Rxn 14: no template NPPA
Rxn 15: no template GAPD (reference gene)
PLATE LAYOUT

Variation 1
Figure 1
Variation 2
Figure 1
Both Variations (1 and 2) are correct. Choose a format that helps you to easily keep track of the samples. If you have a large experiment, try to fit as many reactions on the plate as you can (to avoid wasting plates), but also keep the samples arranged in an orderly fashion so that the set-up won't confuse you.

  • A single plate contains 96 wells. To insure accuracy, three technical replicates per reaction (Rxn) are required
  • If you need more than 96 wells, you must split the experiment over multiple plates.
  • 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

  • Label one 1.5 mL tube per gene target
  • Make enough PCR master mix for your plate...
    • MPK14 is in Reactions 1, 6, and 11 = 3
    • Replicates per reaction = 3
    • Master mix amount = 3 * 3 + 1 (to allow for pipetting error) = 10
    • The same needs to be done for CBX8, TNF, NPPA, and GAPD in separate tubes.


Reagent (Single well) Gene Target (x10) GAPD (x10)
2x LC480 Probes Master (7.5 μL) 75.0 75.0
20 μM Forward primer (0.3 μL) 3.0 3.0 GAPD primers*
20 μM Reverse primer (0.3 μL) 3.0 ---
10 μM UPL probe (0.3 μL) 3.0 3.0 GAPD UPL probe*
PCR H2O (0.1 μL) 1.0 4.0
Total vol. (8.5 μL) 85.0 85.0

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


Resulting 1.5 mL tubes:

  • MPK14 - 85.0 μL
  • CBX8 - 85.0 μL
  • TNF - 85.0 μL
  • NPPA - 85.0 μL
  • GAPD - 85.0 μL

4. Reaction set-up: master mixes for each Template

  • Typically, you will have only 20 μL of stock cDNA on hand.
  • Make a 1:10 dilution of cDNA by adding 10 μL of the stock cDNA to 90 μL of PCR H2O.
  • Make enough Template master mix for your plate...
    • Treated cells cDNA is in Reactions 1, 2, 3, 4 and 5 = 5
    • Replicates per reaction = 3
    • Master mix amount = 5 * 3 + 1 (to allow for pipetting error) = 16
    • The same needs to be done for templates "untreated cells" and "no template" in separate tubes.
Reagent (Single well) cDNA Template (x16)
1:10 cDNA dilution (2.0 μL) 32.0*
PCR H2O (4.5 μL) 72.0
Total vol. (6.5 μL) 104.0

*For the no template control, use PCR H2O instead of cDNA.

Resulting 1.5 mL tubes:

  • T1 - treated cells cDNA, 84.5 μL
  • T2 - untreated cells cDNA, 84.5 μL
  • T3 - no template control, 84.5 μL



5. Reaction set-up: loading the 96-well plate
The following illustrations use Plate Layout Variation 2...

Pipette 19.5 μL of cDNA Template master mixes into the first well of each 3-well group
Figure 3
Add 25.5 μL of Gene Target master mix to the cDNA. After each addition, mix by gently pipetting up and down 3 - 5 times (without making bubbles). Figure 4
Transfer 15 μL of solution from A1 into A2, and A3. Use a fresh pipette tip to do the same for A4-6, and A7-8. Figure 5
Repeat this procedure for the rest of the plate Figure 2
  • Seal the plate with clear film.
  • The plate is now ready to load into the Light Cycler 480!
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