Presentation on theme: "ABI-7900 RT-PCR machine New User’s Guide -Please use “Normal” ppt view to follow the presentation (not a slide show); make sure you can see “The notes”"— Presentation transcript:
ABI-7900 RT-PCR machine New User’s Guide -Please use “Normal” ppt view to follow the presentation (not a slide show); make sure you can see “The notes” at the bottom of the screen - To gain an access to RT-PCR facility, please send your Request for Internal Services (last page of the presentation) to: - - Request security access to the facility: -book RT-PCR machine and Eppendorf robot on-line: - Create your own folder in “7900 data” using your supervisor name + your name - record your usage of RT-PCR machine, reagents, accessories and Eppendorf robot in PCR Records folder (Laptop) within your supervisor’s folder
Real-Time PCR Facility The Technique Quantitative "real time" PCR (qPCR) measures the accumulation of fluorescent DNA amplification products as they are generated during each cycling step of a polymerase chain reaction. This produces a "real time" plot of fluorescence vs cycle number, where the fluorescence is proportional to the amount of amplified product. Quantitative data are obtained by measuring the "cycle threshold" (Ct) - the cycle number at which fluorescent products are first detected, rather than the final amount of fluorescent product accumulated after a fixed number of cycles as in standard PCR. The higher the amount of initial nucleic acid template in a reaction, the sooner fluorescence is detected and the lower the Ct value which is measured. This enables us to quantify the relative abundance of various DNA target sequences in a sample, or absolute abundance of a target by comparison with appropriate standards. The kinetic data produced during the reaction are very useful for quality control without having to open reaction tubes and analyze their contents. For example, the PCR amplification efficiency can be determined during the initial (exponential) phase of the reaction, and frequently approaches the theoretical maximum of 2n when amplifying small DNA target sequences. Fluorescent "melt curves" obtained by heating the final product can determine if the expected PCR amplicon is contaminated by non-specific amplification products. Applications Diagnostics e.g. detection and quantitation of pathogenic bacteria or virus sequences in tissue or environmental samples. RT-qPCR: measurement of transcript (messenger RNA) abundance in RNA samples after reverse transcriptase (RT) to produce cDNA. Allelic discrimination including detection of single nucleotide polymorphisms. Genomic analysis e.g. measurement of copy number of a transfected plasmid construct or stably integrated transgene per host cell genome. Multiplex sequence detection: simultaneous analysis of two or more target sequences within the same reaction tube. Facilities available The Real-Time PCR Facility was set up in 1997 funded from ARC Research Infrastructure & other sources including Biochemistry Dept. & Science Faculty. The Facility provides staff at the St. Lucia campus with access to state-of-the-art instruments for monitoring fluorescence during the polymerase chain reaction (PCR). We offer advice on how to apply this technology to your research needs, including the different chemistries and reagents/consumables available, primer and probe design, optimization of assays, quantitation methods, and provide training in the use of the instruments and data analysis. PCR plates or tubes and some reagents can be purchased through the Facility. ABI 7900 Sequence Detection System -384 well block and low density arrays attachment (micro fluidic card). Eppendorf epMotion 5075 Robotics System (available for setting up assays for real-time PCR).
A clean room free from any DNA templates is available for diluting primers and probes and for setting up the initial part of the PCR assays (without template). 2 PC computers with software: Primer Express for primer/probe design; SDS (Sequence Detection Software) for analysis of data from real time runs. 2 Macintosh computers with Mac versions of the above software. Please see Bob Simpson/ Michael Nefedov for accessing the data analysis software for use on your own computer. Instrument Booking System An online booking system is available via the public folders in Microsoft Outlook. Please contact Bob Simpson/ Michael Nefedov for information. Detection Chemistries A number of alternative fluorescence chemistries have been devised to detect DNA molecules produced during PCR. All can be used with our instruments. The two chemistries used most frequently in our Facility are: SYBR Green dye. This dye binds to the minor groove of double-stranded DNA, and the SYBR-dsDNA complex emits green fluorescence when excited with light of lower wavelength. This chemistry is relatively cheap, but is not specific and produces fluorescence from all dsDNA amplified including non-target sequences and primer dimers if present. TaqMan fluorogenic probes. TaqMan (named after the computer game PacMan) uses a single-stranded DNA probe of ~15-30 nucleotides (labeled with a fluorogenic dye at the 5' end and a fluorescence quencher at the 3' end) homologous to a target sequence between the PCR priming sites. During the extension phase of PCR, the Taq DNA polymerase encounters the bound probe and cleaves it, hence releasing the detection dye from the effect of the quencher. This results in enhanced fluorescence when the dye is excited with incident light. The extra specificity conferred by the detection probe has advantages for many applications eg. for diagnostic applications where false positives are a problem. Design of PCR primers and TaqMan Probes We recommend the use of the Primer Express software to design PCR primers for SYBR Green, or a matched primer-probe set for TaqMan. The software enables primers/probes to be designed for a standard annealing temperature (usually °C for primers) to enable assays for different targets to be run simultaneously in the same reaction plate. Primer Express software is available for use within the Facility. Sample and set-up requirements Users are responsible for obtaining suitably-purified and diluted genomic DNA or cDNA samples, and setting up their own reaction tubes. Bob Simpson/ Michael Nefedov will give advice on how best to do this and avoid the many pitfalls that can bedevil the uninitiated. In general, you will require: DNA or cDNA samples free of "Taq inhibitor", e.g. typically ~20 ng cDNA / 5 μL Primer solutions, typically 1uM but requires optimization. TaqMan probe (if required), typically 2uM but requires optimization. Master mix kit containing a suitable Taq DNA polymerase and buffer. For consistent results from run to run, we recommend you choose a commercial kit rather than prepare your own, despite this representing the largest single cost for accessing this technology. Consult Bob Simpson/ Michael Nefedov for advice. Reaction tubes/strips/plates.
Analysis of data The Ct value for an assay is related to the abundance of template in that reaction. For the absolute quantitation method, a standard curve of a plot of Ct value against the log of the template concentration allows template concentration for unknown samples to be determined. For the relative quantitation method, which is frequently used for mRNA analysis, the abundance of a transcript of a gene of interest is measured relative to that of an endogenously expressed gene such as actin or rRNA (housekeeping gene). Relative quantitation works best when the genes being compared are amplified with similar PCR efficiencies, but corrections can be made if measured differences in efficiency are known. Bob Simpson/ Michael Nefedov will give advice on appropriate experimental design and data analysis strategies, and how to set up spreadsheets to process data. Contacts For training, contact Bob Simpson/ Michael Nefedov, Molecular Biosciences Building (#76) room 341: ('phone , Please follow this link to the Instrument Booking Booking System Charges for use A small charge is made to pay for maintenance of the equipment: ABI 7900 Sequence Detection System: $55 per run SCMB members (384 wells or part thereof) Note: the consumable cost for reagents are in addition to the above costs. Eppendorf epMotion 5075 Robotics System: no charge if used to set up for the above instruments. "Troubleshooting and method development" policy: if technical problems arise during a run requiring a repeat run, or if a series of runs is required to develop protocols/solve problems of general interest to other users, we will reduce or waive the run cost. Consult Bob Simpson/ Michael Nefedov for details.