1. Dr. Soupsana P. Katerina Ioannina, 5/7/13

2. What is Real-time PCR? Real-time PCR is the continuous collection of fluorescent signal from one or more polymerase chain reactions over a range of cycles. Quantitative real-time PCR is the conversion of the fluorescent signals from each reaction into a numerical value for each sample.

3. Where can I use this method? Gene expression analysis Mutation analysis (genotyping) DNA methylation Mitochondrial DNA analysis Real time immuno-PCR Clinical microbiology and virology Hematology Solid organ transplant monitoring Prenatal diagnosis of monogenic diseases caused by single nucleotide changes

4. Real time PCR Instruments Eppendorf Realplex Stratagene Mx3005p Qiagen Rotor-Gene Q Bio-Rad CFX96

5. Roche LightCycler 480 Applied Biosystems 7500 Fast BiomarkOpenArray Real time PCR Instruments

6. High throughput platforms 96  96 (=9216 reactions) Dynamic Array Open Array 3072 reactions Assays Samples

7. Roche Lightcycler

8. Real time PCR: an overview

9. One or two step RT-PCR?

10. ProcedureAdvantages One-stepEasy-Fast Low contamination risk High reproducibility Two-StepMultiple PCRs from a single RT reaction Flexibility with RT primer choice Long-term storage of cDNA Useful when your RNA is in low quantity

11. RNA quality and quantity Bioanalyzer-RIN number/Nanodrop

12. Common used dyes

13. SYBR green method

14. LUX (light upon extension) primers

15. Plexor system Use of isomers of the bases cytodine and guanine, iso-C and iso-G. Iso-dGTP coupled to a dark quencher dye (DABSYL) Signal goes down instead of up!

16. Taqman probes

17. Molecular beacons

18. FRET probes

19. Melting curve analysis

20. Standard curve

21. Amplification Plot

22. Analyze your data

23. Choosing a gene for normalization

24. ΔCt and ΔΔCt method