# Haynes:UPLassay

(Difference between revisions)
 Revision as of 21:16, 17 May 2012 (view source)← Previous diff Revision as of 21:19, 17 May 2012 (view source)Next diff → Line 143: Line 143: '''Reaction Set-up: loading the 96-well plate'''
- Each well will have a total volume of 15.0 μL. How do we end up with that number? + 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, Loading ctrl gene D, treated cDNA, untreated cDNA, and no template. * 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. - * She will pipette 19.5 μL (3 x 6.5) of '''treated cDNA''' dilution to the PCR mix in A1. + * 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 into well A1, and mix by gently pipetting up and down 3 - 5 times (without making bubbles). + * 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. * 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. * She will use the same pipette tip to transfer 15 μL of solution from A1 into A2, and A3. Line 208: Line 208: | H | H |} |} + + The plate is now ready to load into the Light Cycler 480!

## Revision as of 21:19, 17 May 2012

Universal Probe Library Assay
Based on the Universal Probe Library Assay Quick Guide from Roche

• 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.

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:

 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 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 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

Reaction Set-up: template cDNA dilutions

• 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
• 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 is 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

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, Loading ctrl gene D, 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!