For life science research only. Not for use in diagnostic
procedures. FOR IN VITRO USE ONLY.
Universal ProbeLibrary Set,
Mouse
Version April 2009 Set of 90 pretested probes for quantification of gene expression levels by real-
time PCR
Cat. No. 04 683 641 001For 250 × 50 ?l-reactions
(or 625 × 20 ?l-reactions) per probe
Store the probe vials at ?15 to ?25°C
N Keep away from light!
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Table of Contents
1.What this Product Does____________________________________________________________ 3
Number of Tests3 Set Contents3 Storage and Stability 3 Additional Equipment and Reagents Required 4 Application 5 Assay Time 5 2.How to Use this Product __________________________________________________________6 2.1Before You Begin 6
Precautions 6 Sample Material 6 Negative Control7 Primers 7 Universal ProbeLibrary Probes 7 Enzymes, Buffers, and Solutions 7 Passive Reference Dye 8 2.2Real-time PCR protocol for use with LightCycler? 480 System9
Preparation of the PCR Mix 9 LightCycler 480 Instrument Protocol for 96-well Plates 10 LightCycler 480 Instrument Protocol for 384-well Plates 10 2.3Real-time PCR Protocol for use with LightCycler? Carousel-Based System 11
Fluorescence and Run Setup Parameters 12 Preparation of the Master Mix 12 Preparation of the PCR Mix 13 2.4PCR Protocol for use with other Real-time PCR Instruments 14
General Remarks 14 Preparation of the PCR Mix 14 PCR Protocol 15
2.5Assay Design Workflow 16
3.Troubleshooting _________________________________________________________________17
Amplification of genomic DNA17 4.Additional Information on this Product ____________________________________________18
How this Product Works 18 Universal ProbeLibrary Principle 18 References 19 Quality Control 19 5.Supplementary Information ______________________________________________________20 5.1Conventions 20
Text Conventions 20 Symbols 20 5.2Abbreviations20 5.3Changes to Previous Version 20 5.4Ordering Information 21 5.5Disclaimer of License 23 5.6Trademarks23P R O T O C O L
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1.What this Product Does
Number of Tests Each vial of the Universal ProbeLibrary Set, Mouse contains enough probe for 125 assays in a 50 ?l or 312 assays in a 20 ?l PCR setup.
Set Contents The Universal ProbeLibrary Set, Mouse contains 90 vials which are labeled according to the table below:
Each vial contains 125 μl of a 10 μM solution of a pretested probe.
?The Universal ProbeLibrary Set, Mouse is a selection of 90 probes from the
165 available Universal ProbeLibrary probes, specific for the detection of the
mouse transcriptome.
?You can order each probe of the Universal ProbeLibrary Set, Mouse as a
separate single-probe vial containing enough probe for 250 × 50?l-reac-
tions. For further details, visit https://www.doczj.com/doc/967545829.html,.
?Universal ProbeLibrary probes are labeled with fluorescein (FAM) at the 5′-
end and with a dark quencher dye near the 3′-end. The probes can be
detected using standard FAM or SYBR Green I filters.
Storage and Stability Store the set at ?15 to ?25°C through the expiration date printed on the label. For short-term storage (up to 1 month) the probes may be stored at +2 to +8°C.
?The set is shipped on dry ice.
?Keep the probe vials away from light!
?Avoid repeated freezing and thawing. After the first thawing, store the probes aliquoted.
Probe #15Probe #16Probe #17Probe #18Probe #19Probe #20
Probe #31Probe #32Probe #33Probe #34Probe #38Probe #40 Probe #41Probe #42Probe #45Probe #46Probe #47Probe #48 Probe #49Probe #50Probe #51Probe #52Probe #53Probe #55 Probe #66Probe #67Probe #68Probe #69Probe #70Probe #71 Probe #78Probe #79Probe #80Probe #81Probe #82Probe #83 Probe #84Probe #85Probe #88Probe #89Probe #91Probe #92 Probe#93Probe#94Probe#95Probe#96Probe#97Probe#98 Probe #105Probe #106Probe #107Probe #108Probe #109Probe #110
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Additional Equipment and Reagents Required Additional reagents and equipment required to perform quantitative real-time PCR assays with the Universal ProbeLibrary probes include:
?general laboratory equipment
–nuclease-free, aerosol-resistant pipette tips
–pipettes with disposable, positive-displacement tips
–sterile reaction tubes for preparing master mixes and dilutions
–standard benchtop microcentrifuge
?for first-strand cDNA synthesis
–Transcriptor First Strand cDNA Synthesis Kit*
?for use in combination with the LightCycler? Carousel-Based System:
–LightCycler? 2.0 Instrument*, LightCycler? 1.5 Instrument*, or an lower version
–LightCycler? TaqMan? Master*
–LightCycler? Capillaries*
–LC Carousel Centrifuge 2.0* (optional)
?for use in combination with the LightCycler? 480 System:
–LightCycler? 480 Instrument*
–LightCycler? 480 Probes Master*
–LightCycler? 480 Multiwell Plate 384* or LightCycler? 480 Multiwell Plate 96*
?for use in combination with other commercially available real-time PCR instruments including 96- and 384-well block cycler instruments:
–FastStart TaqMan? Probe Master*
–FastStart Universal Probe Master (Rox)* (for use in combination with real-time PCR instruments requiring a reference dye for normalization)
–ROX Reference Dye* (optional)
?for carry-over prevention (optional)
–LightCycler? Uracil-DNA Glycosylase* (in combination with LightCycler?TaqMan? Master or FastStart TaqMan? Probe Master)
–Uracil-DNA Glycosylase, heat-labile* (in combination with other real-time PCR reagents)
–The Universal ProbeLibrary Set, Mouse is also compatible to a generic PCR reaction setup. In this case, Taq DNA polymerase with reaction buffer, MgCl2, and dNTPs are needed. Use of a hot-start Taq DNA polymerase is recommended.
* available from Roche Applied Science
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Application The Universal ProbeLibrary Set, Mouse is intended for life science research. It enables rapid and flexible quantification of virtually any transcript in the mouse
transcriptome by real-time PCR assays. Performance of the assay with the
selected Universal ProbeLibrary probe follows established real-time PCR pro-
tocols using the LightCycler? TaqMan? Master on the LightCycler? Carousel-
Based System, the LightCycler? 480 Probes Master on the LightCycler? 480
System, or the FastStart TaqMan? Probe Master (or other real-time PCR com-
patible reagents) on other real-time PCR instruments.
Gene specific expression quantification assays are easily designed using the
web-based ProbeFinder software accessible at https://www.doczj.com/doc/967545829.html,
or via the Roche Applied Science home page, https://www.doczj.com/doc/967545829.html,.
Assay Time A complete Universal ProbeLibrary assay, including assay design and real-time PCR reaction, needs approx. 2 days. The table below describes the basic
workflow of a Universal ProbeLibrary assay:
Day 1
Decide on target sequence of interest, e.g., entire transcript, exon, or other
sequence.
Identify probes and primers using the ProbeFinder software.
Order primers from your preferred oligo supplier for overnight delivery.
Day 2
Pick the relevant probe from the Universal ProbeLibrary Set and combine it
with the corresponding primers.
Perform real-time PCR.
Evaluate results.
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2.How to Use this Product
2.1Before You Begin
Precautions To reduce the risk of contaminating your PCR reaction with PCR amplicons generated in previous reactions (and consequently false results), as well as of
degrading template nucleic acid, follow the recommendations below:
?Always wear a clean lab-coat. Use separate lab-coats, when setting up PCR
reaction and handling PCR products.
?Change gloves whenever you suspect they have been contaminated.
?Maintain dedicated areas for PCR setup, PCR amplification, and gel electro-
phoresis of PCR products.
?Always use nuclease-free reagents, buffers and consumables.
?Never bring amplified PCR products into the PCR setup area.
?Open and close all sample tubes carefully. Pipet carefully and do not splash
or spray PCR samples.
?Keep reactions and components capped whenever possible. Always spin
tubes before opening.
?Use pipette tips with aerosol filter inserts to avoid aerosol mediated contami-
nation of your pipetting device.
?Clean laboratory benches and equipment regularly.
?Prevent carry-over contamination by using dUTP and Uracil-DNA N-Glyco-
sylase.
Sample Material?Before setting up a Universal ProbeLibrary assay, RNA must be converted to cDNA in a reverse transcription reaction. The quality of RNA is essential for
accurate and reproducible results. Thus, the quality of total RNA should be
high and free of contaminating DNA, RNases and inhibitors. For reproducible
isolation of nucleic acids use:
–either the MagNA Pure LC Instrument or the MagNA Pure Compact
Instrument together with a dedicated nucleic acid isolation kit (for auto-
mated isolation) or
–a High Pure nucleic acid isolation kit (for manual isolation).
For details see the Roche Applied Science Biochemicals catalog or home
page, https://www.doczj.com/doc/967545829.html,.
–Whenever non-intron-spanning assays are performed, the use of DNase-
treated RNA preparations is obligatory.
–The quality of total RNA may be checked by gel electrophoresis to verify
that rRNA is non-degraded and therefore present on the gel as bands.
OD260/280 measurements should also be performed to estimate RNA con-
centration. High quality RNA should have OD260/280 ratios of 1.8 – 2.0
–For storage of template RNA precipitate the RNA with ethanol and keep
the RNA at ?20°C or lower.
?As a guideline 10 ng of total RNA is adequate for detection of a medium
abundant mRNA.
–For initial experiments, run undiluted, 1:10 diluted, and 1:100 diluted cDNA
template in parallel to determine the optimum template amount. A too
high template amount might inhibit the PCR reaction.
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Negative Control Always run negative controls with the samples. To prepare negative controls:?replace the template RNA with PCR-grade water (this will reveal whether a
contamination problem exists)
?omit addition of reverse transcriptase to the cDNA synthesis reaction (this
will indicate whether DNA in RNA samples cause false-positive results)
Primers Use PCR primers at a final concentration of 200 – 900 nM. The recommended starting concentration is 400 nM each. The primer concentration may be opti-
mized by varying the final concentration from 200 - 900 nM in increments of
100 nM.
?To assure optimum performance of your Universal ProbeLibrary assay,
always use highly purified (e.g., HPLC purified) PCR primers.
?Optimize the primer concentration first, then determine the probe optimiza-
tion using the optimized primer concentrations.
?The optimal primer concentration is the lowest concentration that results in
the lowest CP (or Ct) and an adequate fluorescence for a given target con-
centration.
Universal ProbeLibrary Probes Use the probes at a final concentration of 200 nM. Set the annealing tempera-ture during PCR to 60°C.
?The Universal ProbeLibrary probe concentration may be optimized by varying the final concentration from 100 to 500 nM in increments of 100nM.
?The optimal probe concentration is the lowest concentration that results in the lowest CP (or Ct) and an adequate fluorescence for a given target con-centration.
Enzymes, Buffers, and Solutions If you are not using the ready-to-use LightCycler? TaqMan? Master, LightCycler?480 Probes Master, or FastStart TaqMan? Probe Master from Roche Applied Science, already optimized for use of real-time PCR hydrolysis probe assays, the following additional reagent concentrations are recom-mended:
?thermostable DNA polymerase: 2.5 U per 50 ?l reaction
N Use of a hot-start Taq DNA polymerase is recommended.
?final concentration of Mg2+: 3.5 mM
?final concentration of dNTPs (dATP, dCTP, dGTP, dTTP): 200 ?M each
N In case you want to include dUTP into the dNTP mix to perform carry-over prevention with UNG, replace dTTP by 600 ?M dUTP. Increase the final concentration of Mg2+ to 5.5 mM.
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Passive Reference Dye Many real-time PCR instruments will only produce reliable results when a pas-sive reference dye such as Rox is added to the PCR reaction. It is recom-mended to examine whether your real-time PCR instrument has this requirement.
L The LightCycler? Instruments do not require addition of a passive refer-ence dye to the PCR reaction.
The amount of Rox reference dye to include in the PCR reaction depends on the requirements of the real-time PCR instrument and must be adjusted accordingly. However, note that the optimal concentration of passive reference dye is very instrument dependent. Therefore, check the Operator’s Manual of your real-time PCR instrument for this information and read the package inserts of the FastStart TaqMan? Probe Master and the Rox Reference Dye for further details.
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2.2Real-time PCR Protocol for use with LightCycler? 480 System
Preparation of the PCR Mix Follow the procedure below to prepare one 20 ?l standard reaction.
L Do not touch the surface on the LightCycler? 480 Multiwell Plate when handling it.
To prepare the PCR mix for more than one reaction, multiply the amount in the “Volume” column above by the number of reactions to be run and add one additional reaction.
??Thaw the solutions and briefly spin vials in a microcentrifuge before opening.
?Mix carefully by pipetting up and down and store on ice.
?Prepare the PCR Mix for one 20 ?l reaction by adding the following com-ponents in the order listed below:
Component Conc.Vol.Final Conc.
Primer (GOI) forward20 ?M0.4 ?l400nM
Primer (GOI) reverse20 ?M0.4 ?l400nM
UPL probe (GOI)10 ?M0.4 ?l200 nM
LightCycler? 480 Probes Master2×10.0 ?l1×
Water- 3.8 ?l-
T otal Volume15.0 ?l
??Mix carefully by pipetting up and down. Do not vortex.
?Pipet 15 ?l RT-PCR mix into each well of the LightCycler? 480 Multiwell
Plate.
?Pipet 5 ?l Template (cDNA) to the RT-PCR mix of each well.
?Seal Multiwell Plate with LightCycler? 480 Sealing Foil.
?Centrifuge for 2 min at 1,500 × g in a standard swing-bucket centrifuge, containing a rotor for multiwell plates with suitable adaptors.
?Load the Multiwell Plate into the LightCycler? 480 Instrument.
?Start the PCR program described below, depending on whether you use 96-well or 384-well plates.
N If you use reaction volumes different from 20 ?l, be sure to adapt the right volume in the running protocol. As a starting condition, we rec-
ommend to use the same hold times as for the 20 ?l volume.
Real-time PCR Protocol for use with LightCycler
?
480 System
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LightCycler ? 480 Instrument
Protocol for 96-well Plates
The following table shows the PCR parameters that must be programmed for a
LightCycler ? 480 Instrument.?Pre-Incubation for activation of FastStart Taq DNA Polymerase and denatur-ation of the DNA ?Amplification of the target cDNA
?Cooling the rotor and thermal chamber
For details on how to program the experimental protocol, see the LightCycler ?480 Operator’s Manual.
L For details on how to program the experimental protocol, see
the LightCycler ? 480 Instrument Operator’s Manual.
LightCycler ? 480
Instrument
Protocol for 384-well Plates
Filters : Please use the channel FAM / SYBR Green I (Emission: 533 nm)
for UPL FAM-Assay.
Analysis Mode
Cycles
Segment
Ramp rate / Slope (°C/s)Target
Temp.Time
Acquisition Mode
Pre-Incubation
None
1
- 4.495°C
10 min None
Amplification
Quantifica-tion
45Denatur-ation 4.4
95°C 10 s None Annealing 2.2
60°C 30 s None Extension
4.472°C 1 s Single Cooling None
1
2.2
40°C
30 s
None
Analysis Mode
Cycles Segment
Ramp rate / Slope (°C/s)Target
Temp.Time
Acquisition Mode
Pre-Incubation
None
1
- 4.895°C 10 min None Amplification
Quantifica-tion
45Denatur-ation 4.8
95°C 10 s None Annealing 2.5
60°C 30 s None Extension
4.872°C 1 s Single Cooling
None
1
2.5
40°C
30 s
None
R e a l -t i m e P C R P r o t o c o l f o r u s e w i t h L i g h t C y c l e r ? 480 S y s t e m
2.2Real-time PCR Protocol for use with LightCycler ? 480 System, continued
2.3Real-time PCR Protocol for use with LightCycler Carousel-Based System
LightCycler?Carousel-Based System The following procedure is optimized for use with the LightCycler? 1.5, 2.0 Instruments or lower versions.
N Program the LightCycler? Instrument before preparing the reaction mixes.
The protocol for a Universal ProbeLibrary assay using the LightCycler?TaqMan? Master consists of the following parts:
?UNG Incubation (optional; only needed if carry-over prevention using LightCycler? Uracil-DNA Glycosylase is performed).
?Pre-Incubation for activation of FastStart Taq DNA Polymerase and dena-turation of the DNA
?Amplification of the target cDNA
?Cooling the rotor and thermal chamber
For details on how to program the experimental protocol, see the LightCycler?Operator’s Manual for the respective instrument.
L Temperature Transition Rate/Slope is 20°C/sec.
N Set all other protocol parameters not listed in the tables below to ‘0’.
The following table shows the PCR parameters that must be programmed for a LightCycler? Carousel-Based System PCR run with
the LightCycler?TaqMan? Master.
Analysis
Mode
Cycles
Target
Temp.
Time
Acquisition
Mode
UNG Incubation (optional)
None140°C 2 min None
Pre-Incubation
None195°C10 min None
Amplification
Quantification4595°C10 s None
60°C 30
s None
72°C 1 s Single
Cooling
None140°C30 s None
LightCycler
?
Carousel-based System Real-time PCR Protocol Real-time PCR Protocol for use with LightCycler
?
Carousel-Based System
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Fluorescence and
Run Setup
Parameters Preparation of the Master Mix
Prepare the 5× Master Mix as described below:Parameter Setting Seek Temperature
30°C
Default Channel
?during run ?for analysis ?fluorescence channel 530 and 560
(530 and 640 for the LightCycler ? 1.5 Instrument or lower version)Fluorescence Gains
not required
“Max. Seek Pos”Enter the number of sample positions the instrument should look for.
“Capillary Size”
Select “20 ?l” as the capillary size for the experiment.
?Thaw one vial of “Reaction Mix” (vial 1b, red cap).
?Briefly centrifuge one vial “Enzyme” (vial 1a, white cap) and the
thawed vial of “Reaction Mix” (from Step 1).?Pipet a total volume of 10 ?l from vial 1a (“Enzyme”, white cap) into
vial 1b (“Reaction Mix”, red cap). (One vial 1a contains enzyme for 3vials Reaction Mix.)?Mix gently by pipetting up and down.
N Do not vortex.
R e a l t -t i m e P C R P r o t o c o l f o r u s e w i t h L i g h t C y c l e r ? C a r o u s e l -B a s e d S y s t e m
Preparation of the PCR Mix Proceed as described below for a 20 ?l standard reaction.
N Do not touch the surface of the capillaries. Always wear gloves when han-dling the capillaries.
3Depending on the total number of reactions, place the required num-ber of LightCycler? Capillaries in precooled centrifuge adapters or in a
LightCycler? Sample Carousel in a precooled Centrifuge Bucket from
the LC Carousel Centrifuge 2.0.
opening.
?Mix carefully by pipetting up and down and store on ice.
?Prepare 20 ?M solutions of the PCR primers.
?Prepare the PCR Mix for one 20 ?l reaction by adding the following components in the order mentioned below:
Component Conc.Volume Final conc.
Primer (GOI) forward20 ?M0.4 ?l400nM
Primer (GOI) reverse20 ?M 0.4
?l400nM UPL probe (GOI)10 ?M0.4 ?l200nM
LightCycler TaqMan Master, (vial 1,
red cap)
5× 4.0 ?l1×
Water, PCR-grade (vial 2, colorless
cap)
9.8 ?l
Total volume15.0 ?l
L To prepare the PCR mix for more than one reaction, multiply the amount in the “Volume” column above by the number of reactions
to be run and add one additional reaction.
′?Mix carefully by pipetting up and down. Do not vortex.
?Pipet 15 ?l PCR mix into each precooled LightCycler? Capillary.
?Add 5 ?l of the cDNA template.
?Seal each capillary with a stopper.
top microcentrifuge.
?Centrifuge at 700× g for 5 s (3,000 rpm in a standard benchtop micro-
centrifuge).
?Alternatively, use the LC Carousel Centrifuge for spinning the capil-
laries.
?Transfer the capillaries into the sample carousel of the LightCycler?Instrument.
oCycle the samples as described above.
Real-time PCR protocol for use with LightCycler
?
Carousel-Based System
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2.4PCR Protocol for use with other Real-time PCR Instruments
General Remarks FastStart Universal Probe Master (Rox) generates excellent results on instru-ments such as the Applied Biosystems 7900HT Real-Time PCR System or the Applied Biosystems 7500 Real-Time PCR System or all other instruments that require mormalization with Rox.
The FastStart TaqMan ? Probe Master does not contain a Rox reference dye and is therefore the reagent of choice for all instruments that do not require Rox besides the LightCycler ? Instruments.
L If you plan to supplement the FastStart TaqMan ? Master mix with the Rox
reference dye, we recommend using a 1 mM stock solution of Rox*.
Preparation of the PCR Mix
Follow the procedure below to prepare one 20 ?l standard reaction..To prepare the PCR mix for more than one reaction, multiply the amount in the “Volume” column above by the number of reactions to be run and add one additional reaction.
The following table shows the PCR parameters that must be programmed according to the Operator’s Manual of the respective instruments.
3?Thaw the solutions and, to ensure recovery of all the contents, briefly
spin vials in a microcentrifuge before opening.
?Mix carefully by pipetting up and down and store on ice.
?Prepare the PCR Mix for one 20 ?l reaction by adding the following
components in the order listed below:
Component Conc.Volume
Final Conc.Primer (GOI) forward 20 ?M 0.5 ?l 500 nM Primer (GOI) reverse 20 ?M 0.5 ?l 500 nM
UPL probe (GOI)
10 ?M
0.5 ?l
250 nM Mastermix [FastStart Universal Probe Master (Rox) or FastStart TaqMan ? Probe Master 2 ×10.0 ?l 1
×
Water -
3.5 ?l -
Total volume
15.0 ?l
??Mix the solution carefully by pipetting it up and down. Do not vortex.
?Pipet the 15 ?l RT-PCR mix into reaction vessel or well of a PCR microplate (depending on your real-time PCR instrument).?Pipet 5 ?l of template (cDNA) to the RT-PCR mix of each well.?According to the instructions supplied with your instrument, prepare the
tubes or microplates for PCR (e.g ., seal with appropriate caps or self-adhesive foil).?Start the PCR program described below.
P C R P r o t o c o l f o r u s e w i t h o t h e r R e a l -t i m e P C R I n s t r u m n e t s
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PCR Protocol
N For best results, be sure the instrument is correctly calibrated. Set the
detection channel of your real-time PCR instrument to either SYBR Green I or FAM (i.e., 530 nm) for detection of UPL probes labeled with FAM. For the Universal ProbeLibrary reference gene assays, use the VIC/HEX chan-nel (or the next possible emission filter moving to longer wavelengths, e.g .560 nm or 568nm).
?UNG Incubation (optional) only needed if carry-over prevention using LightCycler ? Uracil-DNA Glycosylase is performed.
?Pre-Incubation for activation of FastStart Taq DNA Polymerase and dena-turationof the DNA
?Amplification of the target cDNA.L Please note : when performing UPL assays on Applied Biosystems’ Fast
Real-time PCR Systems, the use of the Fast Mode protocol may generate sub-optimal results.
3
Following the instruction manual of the instrument supplier, program the instrument with the following parameters:
Analysis
Mode Cycles
Target
Temperature Time
UNG Incubation (optional)
1
(optional)
None
50°C 2 min
Pre-Incubation
1None 95°C 10 min
Amplification
45
Quantification
Follow the instructions of your real-time PCR instrument supplier.
·Place your tubes or plate in the instrument and start the reaction.
?
At the end of the reaction, follow instrument instructions for quantification/analysis.
2.4PCR Protocol for use with other Real-time PCR Instruments, continued
PCR Protocol for use with other Real-time PCR Instruments
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2.5Assay Design Workflow
The selection of Universal ProbeLibrary probes and the design of primers for real-time PCR is made using the web-based ProbeFinder software available at the Universal ProbeLibrary Assay Design Center at https://www.doczj.com/doc/967545829.html,.
L The ProbeFinder software has an extensive help function that may be vis-ited whenever in doubt.L For a more detailed description of the ProbeFinder software, incl. advanced options
like assays that target specific exons or splice variants, visit https://www.doczj.com/doc/967545829.html, or read the Universal ProbeLibrary Guide to Successful qPCR Assays.
3?Open your web browser and go to https://www.doczj.com/doc/967545829.html,.
?Select Assay Design Center from the navigation bar on the left side of the screen.
?On the Assay Design Center start screen select your organism of interest.·?On the subsequent page submit a sequence string or a sequence ID.
?Choose whether you want to design an intron-spanning assay: tick the “Automatically select an intron spanning assay” option.
?For dual-color assays choose dual-color assays and one of the refer-ence gene assays or select “Any”.?Click the “Design” button.??The software will select the best assay, which subsequently is pre-sented on the Result Screen (together with the best suitable refer-ence gene assay).?? If for some reason the designed assay is different from what you
desire, alternative and often just as efficient assays are available upon clicking the “More Assays”.
3.Troubleshooting
L For further troubleshooting hints read the detailed instructions of the real-
time PCR reagents used (e.g. Light-Cycler? 480 Probes Master, or the
FastStart Universal Probe Master).
Amplification of genomic DNA The option “Automatically select an intron spanning assay” forces ProbeFinder to find probes near exon-exon splice junctions. Nevertheless, keep in mind that remaining genomic DNA can still be coamplified during the PCR: the risk is higher the shorter an intron is. To avoid this problem use only template RNA for cDNA synthesis that is of high quality and free of any contaminating DNA. Additionally, you might lower the extension time during PCR (down to 10 s) to reduce the risk of amplifying intron sequences.
Cause Recommendation
No fluorescent signal detected during the PCR.Confirm that you have a PCR product by running an aliquot of your PCR reaction on an agarose gel.
No fluorescent signal detected during the PCR and no PCR amplicon can be detected by aga-rose gel electro-phoresis??Insufficient template
amount.
?Inappropriate PCR
parameters.
?PCR inhibition.
?Add more cDNA if you are working with a
very low abundance transcript.
?Optimize the real-time PCR conditions (e.g.,
MgCl2 concentration or addition of T m
decreasing reagents) until you get a PCR
product.
?Repeat isolation of your template.
No fluorescent signal detected during the PCR, but a PCR ampli-con can be detected by aga-rose gel electro-phoresis?Wrong detection channel or
parameters.
?Check that the detection channel in the
real-time PCR cycler was set to either SYBR
Green I or FAM (i.e., 530 nm).
?Check that the optical read is at the correct
step of the real-time PCR cycles.
?Adjust the baseline in the real time PCR
cycler software.
?Perform a real-time PCR with a positive
control.
Weak fluores-cence signals or oddly shaped amplification curves.Inappropriate concentration of
reference dye.
Confirm that the fluorescence signal from
the reference dye is not significantly higher
than the background fluorescence from the
Universal ProbeLibrary probe (i.e., the fluo-
rescence level before reaching the CP or
Ct). Adjust the amount of reference dye
accordingly.
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4.Additional Information on this Product
How this Product Works The Universal ProbeLibrary probes enable quantification of gene expression levels of a large number of organisms (human, primates, mouse, rat, C. ele-gans, Drosophila, Zebrafish, Rice, Maize, Yeast, Arabidopsis) and other organ-isms included in the NCBI Reference Sequence Database.
The Universal ProbeLibrary applies the hydrolysis probe format for real-time PCR detection. Hydrolysis probes contain two labels in close proximity to each other: a fluorescent reporter dye at the 5′-end and a (fluorescent or dark) quencher label at or near the 3′-end. When the probe is intact, the fluorescent signal is almost completely suppressed by the quenching label. When the probe is hybridized to its target sequence, it is cleaved by the 5′ 3′ exonucle-ase activity of the FastStart Taq DNA Polymerase, which "unquenches" the flu-orescent reporter dye. During each PCR cycle, more of the released fluorescent dye accumulates, boosting the fluorescent signal.
Universal ProbeLibrary Principle The specificity of the PCR assay is determined by a combination of the PCR primers, that specify the exact target amplicon among billions of nucleotides, and the probe. The unique universality of Universal ProbeLibrary has been achieved by shortening the length of the dual-labeled probes to only 8-9 nucleotides. The specificity, T m and compatibility are maintained by inclusion of the duplex stabilizing DNA analogue LNA (Locked Nucleic Acid) in the probes (2). The sequences of the probes have been carefully selected to ensure optimal coverage of all transcripts in the respective organism tran-scriptome and a high degree of specificity (3, 4, 5). The selection of Universal ProbeLibrary probes and the design of primers for real-time PCR is made with the ProbeFinder assay design tool, available at https://www.doczj.com/doc/967545829.html,. The Universal ProbeLibrary reference gene assays have 12mer probes that are partially modified with LNA. By performing a dual color assay the ProbeFinder software designs first a Universal ProbeLibrary assay for the gene of interest (GOI) and secondly select the best suitable reference gene assay from a list for the GOI.
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4.Additional Information on this Product, continued
References1Bustin, SA (2000). Absolute quantification of mRNA using real-time reverse tran-
scription polymerase chain reaction assays. J. Mol. Endocrinology25:169-93.
2Holland PM et al (1991). Detection of specific polymerase chain reaction product
by utilizing the 5′→ 3′ exonuclease activity of Thermus aquaticus DNA
polymerase. Proc. Natl. Acad. Sci. USA. 88, 7276-7280.
3Petersen, M and Wengel, J (2003). LNA: a versatile tool for therapeutics and
genomics. Trends Biotechnol. 21, 74-81.
4Mouritzen P et al. (2004). The ProbeLibrary - Expression profiling 99% of all
human genes using only 90 dual-labeled real-time PCR Probes. Biotechniques
37, 492-495.
5Mouritzen, P et al (2005). ProbeLibrary: A new method for faster design and
execution of quantitative real-time PCR. Nature Methods2, 313-17.
6Mauritz, RP et al (2005). Universal ProbeLibrary Set: One Transcriptome – One
Kit. Biochemica2, 22-24.
7Rees, E et al. (2005). Gene Silencing Using esiRNA - Efficient, Robust, and Not
Influenced by Positional Effects. Biochemica3, 26-28.
8Steckel, M and Boutros, M (2005). Rapid Development of Real-Time RT-PCR
Assays Using Universal ProbeLibrary: Applications for Dissecting Signaling Path-
ways by RNA Interference. Biochemica 3, 17-19.
9Kruhoefer et al. (2005). Gene expression signatures for colorectal cancer micro-
satellite status and HNPCC. British Journal of Cancer92, 2240-2248.
10Magnusson et al. (2005). Gene expression changes induced by bismuth in a
macrophage cell line. Cell Tissue Res321, 195-210.
Quality Control?All UPL probes have passed a real-time PCR performance test.
?Each UPL probe is analyzed by anion-exchange HPLC and MALDI-MS to
ensure purity and quality.
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5.
Supplementary Information
5.1
Conventions
Text Conventions
To make information consistent and memorable, the following text conventions are used in this Instruction Manual:Symbols
In this Instruction Manual, the following symbols are used to highlight important infor-mation: 5.2
Abbreviations
In this Instruction Manual, the following abbreviations are used:
5.3
Changes to Previous Version
?editorial changes
?real-time PCR protocols improved
Text Convention Usage
Numbered stages labeled ?, ?, etc.Stages in a process that usually occur in the order listed.
Numbered instructions labeled 3, ?, etc.Steps in a procedure that must be performed in the order listed.
Asterisk *
Denotes a product available from Roche Applied Sci-ence.
Symbol
Description
L Information Note:
Additional information about the current topic or procedure.
N
Important Note:
Information critical to the success of the procedure or use of the product.
Abbreviation Meaning CP crossing point Ct
threshold cycle
GOI
Gene of Interest
qPCR
quantitative real-time PCR
RG
Reference Gene
UNG
Uracil-DNA Glycosylase
UPL
Universal ProbeLibrary