dr. soupsana p. katerina ioannina, 5/7/13. what is real-time pcr? real-time pcr is the continuous...

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