Patrick Maaß, B.Sc., wurde 1973 in Wattenscheid geboren. Nach einer Ausbildung zum Biologielaboranten bei einem großen deutschen Pharmaforschungsunternehmen entschied sich der Autor, seine fachlichen Qualifikationen durch ein Studium weiter auszubauen. Das Studium der Biologie schloss er im Jahr 2009 an der Johannes-Gutenberg-Universität erfolgreich mit dem akademischen Grad des Bachelor of Science (Fachrichtung Molekularbiologie) ab.

Multiplexing of PCR Assays 
1 
Table of Contents 
3 
1 Introduction 
4 
1.1 Breast Cancer 
4 
1.2 RNA Expression Profiling and Prediction 
5 
1.3 Objectives 
6 
2 Materials and Methods 
8 
2.1 Materials 
8 
2.1.1 Instruments and Consumables 
8 
2.1.2 Buffers and Chemicals for Nucleic Acid Purification 
8 
2.1.3 Nucleic Acids, Cell Lines & Tissue Samples
8 
2.1.4 Synthetic Oligonucleotides 
9 
2.1.5 Chemicals and Kits for PCR 
9 
2.1.6 Software 
9 
2.2 Methods 
10 
2.2.1 General Approach 
10 
2.2.2 Purification of Nucleic Acids 
10 
2.2.3 Creation of a Standard Reference RNA Pool 
11 
2.2.4 Real-Time Kinetic RT-PCR 
12 
2.2.5 Designing Dually Labeled Primer-Probe Sets 
13 
2.2.6 Dilution of Primer-Probe Sets 
14 
2.2.7 Setup of Singleplex kPCR Assays 
14 
2.2.8 Evaluation of Different Reporter Dyes 
15 
2.2.9 Controlling of Primer-Probe-Set Performance 
16 
2.2.10 Testing Combinations of Two Primer-Probe Sets (Duplexing) 
17 
2.2.11 Testing Combinations of Three Sets (Triplexing) 
19 
3 Results 
21 
3.1 Identification of Suitable Reporter Dyes 
21 
3.2 Evaluation of Primer-Probe-Performance 
26 
3.3 Identification of Suitable Duplex Combinations 
27 
3.4 Identification of Suitable Triplex Combinations 
30 
4 Discussion 
35 
4.1 Statistical Evaluation of Primer Performances 
35 
4.1.1 Slope 
35 
4.1.2 Efficiency 
36 
4.1.3 Y-Intercept 
38 
4.2 Suitable Reporter Dyes and Quencher 
39 
4.3 Optimization of Assays and kinetic RT-PCR Parameter 
40 
4.4 Conclusion and Outlook 
41 
5 Summary 
43 
6 Literature 
44 
7 Appendix 
47 
7.1 Abbreviations 
47 
7.2 Figures 
48 
7.3 Tables 
49 
7.4 Oligonucleotide Sequences 
50 
7.5 Origins of Breast Cancer Samples for MAVPOOL080623a 
52 
7.6 Acknowledgements 
53 

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).