dr. soupsana p. katerina ioannina, 5/7/13. what is real-time pcr? real-time pcr is the continuous...
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Quantitative Real time PCR Dr. Soupsana P. Katerina
Ioannina, 5/7/13
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.
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
Real time PCR Instruments
Eppendorf Realplex
Stratagene Mx3005p
Qiagen Rotor-Gene Q
Bio-Rad CFX96
Roche LightCycler 480Applied Biosystems 7500 Fast
BiomarkOpenArray
Real time PCR Instruments
High throughput platforms
9696 (=9216 reactions) Dynamic Array
Open Array3072 reactions
Assays
Samples
Roche Lightcycler
Real time PCR: an overview
One or two step RT-PCR?
One or two step RT-PCR?Procedure Advantages
One-step Easy-Fast
Low contamination risk
High reproducibility
Two-Step Multiple PCRs from a single RT reaction
Flexibility with RT primer choice
Long-term storage of cDNA
Useful when your RNA is in low quantity
RNA quality and quantity
Bioanalyzer-RIN number/Nanodrop
Common used dyes
SYBR green method
LUX (light upon extension) primers
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!
Taqman probes
Molecular beacons
FRET probes
Melting curve analysis
Standard curve
Amplification Plot
Analyze your data
Choosing a gene for normalization
ΔCt and ΔΔCt method
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