Textprobe: Kapitel 2.2.4, Real-Time Kinetic RT-PCR: The real-time quantitative RT-PCR method used to quantify RNA in breast cancer tissue combines two successive steps. First, RNA is transcribed into cDNA by the enzyme reverse transcriptase, an RNA-dependent DNA-polymerase, which was first discovered in retroviruses and which is able to synthesize a RNA-DNA-hybrid-strand from a single-stranded RNA, degrade the residual RNA and complete the molecule into a double-stranded cDNA. In the second step, the cDNA serves as a template for the following quantitative polymerase chain reaction (PCR). The PCR uses two sequence-specific oligonucleotides and a DNA-dependent polymerase to amplify a definite DNA segment. An improvement of the PCR is the real-time quantitative PCR, where a third oligonucleotide, a hybridization probe labeled with two different fluorescent dyes and located between the forward- and the reverse-primer, is used. Since one dye works as the reporter dye (i.e. FAM, Cy5, Yakima Yellow, etc.) and the other one as the corresponding quencher (like TAMRA, BHQ1, BHQ2, etc.), the quencher absorbs the emission of the reporter dye by fluorescent resonance energy transfer (FRET). When this dual-labeled probe hybridizes with the template DNA, the 5'-3' nucleolytic activity of the polymerase degrades this probe resulting in a loss of quenching activity. Thus, a continuous increase of occurs during PCR. The amount of fluorescence at a given time point during PCR corresponds to the amount of PCR product. Since fluorescence is measured following each PCR cycle, it is possible to observe the amplification process ,,real-time" and to count back to the initial amount of cDNA. 2.2.5 Designing Dually Labeled Primer-Probe Sets Primer design was accomplished with the help of the software tool Primer Express v2.0.0 from Applied Biosystems. Although this software was built for designing TaqMan® primer and probe sets, it delivers excellent results for other real-time applications such as the MX3005p. When choosing TaqMan® Primer and Probe Design, the software operates with predefined parameters using empirical rules to calculate optimal sequences based upon the input sequence. The most important parameters for the probe were: - Amplicon size should range from 50 - 150 base pairs (bp) - G/C content should be kept between 30% and 80% - Avoiding repeats of identical bases - especially of Guanine - The melting temperature should be between 68°C and 70°C - No 5'-terminal Guanine - Primers should be designed a close to the probe as possible The corresponding forward- and reverse-primer were also automatically designed by that software and their melting temperature should have been about 10°C below the probe-temperature. Although all samples were treated with DNAse, this digestion is very often imperfect. The use of RNA-specific primer probe sets copes with that specific problem. In order to avoid amplification of genomic DNA, RNA-specific, intron-spanning primer-probe sets were designed if possible, based upon the cDNA sequence. All primer-probe sets were ordered from Microsynth, Switzerland in 0.2µmol scale and HPLC purified. 2.2.6, Dilution of Primer-Probe Sets: Each set consisted of two standard oligonucleotides and one dual-labeled probe. Both, the unmodified and the modified oligonucleotides were first diluted to a final concentration of 100µM according to the documents provided by Microsynth. The working solution consisted of 50µl (each) forward and reverse primer and 25µl probe filled up with nuclease free water to a total volume of 1000µl. Thus, the working solution consisted of two unmodified oligonucleotides (5µM each) and one dual-labeled probe (2,5µM). Excerpted from Multiplexing of Pcr Assays in Breast Cancer Analysis by Patrick Maaß All rights reserved by the original copyright owners. Excerpts are provided for display purposes only and may not be reproduced, reprinted or distributed without the written permission of the publisher.
