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UPL Probe Chemistry
Like Taqman probes the UPL probes are hydrolysis probes. Hydrolysis probes contain two labels in close proximity to each other: a fluorescent reporter dye at the 5´-end (FAM) 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´ exonuclease activity of the Taq DNA Polymerase, which "unquenches" the fluorescent reporter dye. During each PCR cycle, more of the released fluorescent dye accumulates, boosting the fluorescent signal.
The Molecular Genetic facility houses a library of 165 tubes, each containing a unique UPL probe. UPL probes are only 8 to 9 nucleotides long. Due to this short size the binding motifs of these probes are very prevalent in many locations of the genome of many organisms. The sequences of the probes have been targeted to bind transcriptomes with higher frequency, ensuring optimal coverage of many genes in many species. Within the human transcriptome, each probe binds to approximately 7000 transcripts, while each transcript is detected by approximately 16 different probes. Only one specific transcript is detected at a time in a given PCR assay, as defined by the set of chosen PCR primers. For each assay, the design software suggests an optimal set of PCR primers, a probe, and any possible alternative sets.
LNA technology used in the UPL probe system
|Figure 1: The UPL probes are only 8 to 9 mers because they use LNAs (Locked Nucleic Acids) which have increased binding strengths compared to standard DNA nucleotides. Locked Nucleic Acids (LNAs) have their ribose ring "locked" with a methylene bridge connecting the 2'-O atom with the 4'-C atom.||Figure 2: LNA nucleosides therefore have a higher annealing temperature compared to standard nucleosides. The locked ribose conformation enhances base stacking and backbone pre-organization, this gives rise to an increased thermal stability and discriminative power of duplexes. LNA discriminates single base mismatches under conditions not possible with other nucleic acids.|
To find the UPL probe for your gene and the sequence of the primers is easy. An online design software will do the job for you. The UPL probe assay design software is accessible online via Roche's Universal Probe Assay Design webpage. This picture shows you the start page:
Just select the organism in the selection field and the following webpage will automatically open:
Enter either the sequence, gene name or ID in this webpage and click on the "Design" button. The assay design software will provide you with a list of UPL probes and primers that will bind to your gene of interest. For example the assay design result for human GAPDH is shown below.
The UPL probe shown in the result window is available in the Molecular Genetics facility's UPL probe library on level 8, Garvan.
The sequence of the primers shown in the result window needs to be copied and pasted into an Primer Order Form and send to our email address. The primers will arrive 2 to 3 days later, you will be notified when they are ready for collection in the facility.
Information on how to setup the realtime PCR reactions is given in the document UPL_Probe_Setup document.
Information on how to set up relative quantative gene expression experiments on the LightCycler 480 (from subsets to meaningful plate layouts) can be found in this pdf-document Relative_Quantification.pdf.
Human Reference Gene Controls
The facility also holds a Human Reference Gene UPL set, which consists of realtime qPCR assays for 10 human reference genes for gene expression quantification in multiplex reactions. These probes are LightCycler® Yellow 555 labelled so that they can be amplified in the same well as the target gene, using dual-color real-time PCR (data collection in FAM and VIC/555 channel). Alternatively they can be amplified in a separate well.
When selecting the multiplex option in the online assay design software, the probe finder software will design multiple include the reference genes into your assay design. At the same time, each assay is subjected to in silico PCR testing to verify that it can be multiplexed with one (or more) of the UPL Reference Gene Assays. This is the list of available human house keeper genes:
- Human G6PD Gene Assay (glucose-6-phosphate-1-dehydrogenase)
- Human PPIA Gene Assay (peptidylprolyl isomerase A / Cyclophilin A)
- Human GAPD Gene Assay (glyceraldehyde-3-phophate dehydrogenase)
- Human TBP Gene Assay (TATAA-box-binding protein)
- Human β2M Gene Assay (β2 microglobulin)
- Human GUSB Gene Assay (β-glucuronidase)
- Human PBGD Gene Assay (porphobilinogen deaminase)
- Human HPRT Gene Assay (hypoxanthine-guanidine phosphoribosyltransferase)
- Human ACTB Gene Assay (β-actin)
- Human PGK1 Gene Assay (phosphoglyceratekinase 1)
For each of the reference genes one vial with 100 µl of a 20 µM solution of two prevalidated primers, and one vial with 100 µl of a 10 µM solution of the reference gene specific probe. All assay tubes are stored in the Reference Gene Box in the facility, please ask facility staff.
Mouse Reference Gene Controls
The facility holds one Roche Mouse Reference Gene UPL assay. This probe is VIC labelled so that it can be amplified in the same well as the target gene, using dual-color real-time PCR (data collection in FAM and VIC channel). There is one tube which contains a gene-specific probe for the mouse ACTB (β-actin) gene at 10 μM (VIC labbeled) and one tube with the primers at 20 μM each.
We also offer a c-alpha based in house reference gene assay which can be used instead of the commercial ACTB assay from Roche. This c-alpha based assay uses primer 1327+1328 (stored as stock concentration of 100 μM in the Reference Gene Box) and a probe called 1329 which is available as Alexa or VIC labelled version (100 μM). Dilute the stocks 1:10 and use 1 ul of each primer and 1 ul of the probe per 10 ul total reaction volume.
Setting Up Your Realtime Experiment
Within the Molecular Genetics facility we keep and maintain the complete UPL Library which you can access at any time and take as much or as little of any of the probes as you need. You will pay by usage (in microlitres) and are asked to fill out the UPL customer log which is located in room 8.05 at the wall near the back entrance. Please record your first and last name, what probe/s were taken (number/s), the date, and indicate how much of each probe was taken (in ul). If you notice that probes are running low please notify us so we can order more.
We also keep stocks of the "Light Cycler 480 Probes Master" Cat# 04 887 301 001 (Roche) which is best used for realtime PCR setup with UPL probes. We sell the necessary 384 well microtiter-plates "Light Cycler 480 Multiwell Plate 384", Cat# 04 729 749 001 (Roche) as well as the Optical Seals (Microseal ‘B’ Film PCR Sealers), Cat#: MSB1001. Please see the Molecular Genetics shop webpage for prices.
There is one LightCycler 480 instrument at the Molecular Genetics facility which can be booked on the booking system "GMG_LC480_1313". If this instrument is overbooked please ask facility staff and we might be able to place your PCR plate on our Mouse Genotyping Service LightCyclers.
The software to setup and analyse the data is called LightCycler 480 software and can be downloaded from Pandora server, path: Pandora/Volumes/Software/Windows/GMG_Software/LightCycler 480 file LightCycler480_Software_Setup.exe. The software only installs on Windows operating system. After installation the login is: Username= admin, password= LightCycler480 (attention capital C in Cycler).
Alternatively you can use the 7900HT or the Biorad C1000 instruments for running your 384 well plate. please ask us for help as the default run parameter need to be changed. For PCR setup in 384 well plates (the facility only holds 384 well PCR instruments) we strongly recommend to use the epMotion 5070 liquid handler robots. How to use the LightCycler 480 and the epMotion robots is explained in individual training sessions on our training ipad which can be booked via the booking system.