Haynes:UPLassay

From OpenWetWare

Revision as of 23:24, 19 January 2013 by Karmella Haynes (Talk | contribs)
Jump to: navigation, search

<- 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 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. 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 need = 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

  • A single plate contains 96 wells (as shown below). 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. Set up your reactions: 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

Tubes:

  • Gene A - untreated cDNA, 84.5 μL
  • Gene B - treated cDNA, 84.5 μL
  • Gene C - no template, 84.5 μL

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

  • 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 Template (x13) untreated cDNA Template (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

Tubes:

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



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 Target A, Gene Target B, Gene Target C, Gene target GAPD, treated cDNA Template, untreated cDNA Template, and no Template.
  • She will pipette 19.5 μL of treated cDNA Template master mix into well A1.
  • She will add 25.5 μL of Gene Target A 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 Template master mix and Gene Target master mix as shown below:
  1 2 3 4 5 6 7 8 9 10 11 12
A treated + A "     "     treated + B "     "     treated + C "     "     treated + GAPD "     "    
B untreated + A "     "     untreated + B "     "     untreated + C "     "     untreated+ GAPD "     "    
C no template + A "     "     no template + B "     "     no template + C "     "     no template + GAPD "     "    
D --- --- --- --- --- --- --- --- --- --- --- ---
E --- --- --- --- --- --- --- --- --- --- --- ---
F --- --- --- --- --- --- --- --- --- --- --- ---
G --- --- --- --- --- --- --- --- --- --- --- ---
H --- --- --- --- --- --- --- --- --- --- --- ---

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

Personal tools