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Quantitative Real time PCR Dr. Soupsana P. Katerina Ioannina, 5/7/13

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Page 1: 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

Quantitative Real time PCR Dr. Soupsana P. Katerina

Ioannina, 5/7/13

Page 2: 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

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.

Page 3: 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

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

Page 4: 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

Real time PCR Instruments

Eppendorf Realplex

Stratagene Mx3005p

Qiagen Rotor-Gene Q

Bio-Rad CFX96

Page 5: 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

Roche LightCycler 480Applied Biosystems 7500 Fast

BiomarkOpenArray

Real time PCR Instruments

Page 6: 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

High throughput platforms

9696 (=9216 reactions) Dynamic Array

Open Array3072 reactions

Assays

Samples

Page 7: 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

Roche Lightcycler

Page 8: 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

Real time PCR: an overview

Page 9: 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

One or two step RT-PCR?

Page 10: 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

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

Page 11: 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

RNA quality and quantity

Bioanalyzer-RIN number/Nanodrop

Page 12: 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

Common used dyes

Page 13: 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

SYBR green method

Page 14: 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

LUX (light upon extension) primers

Page 15: 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

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!

Page 16: 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

Taqman probes

Page 17: 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

Molecular beacons

Page 18: 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

FRET probes

Page 19: 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

Melting curve analysis

Page 20: 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

Standard curve

Page 21: 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

Amplification Plot

Page 22: 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

Analyze your data

Page 23: 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

Choosing a gene for normalization

Page 24: 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

ΔCt and ΔΔCt method


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Real time PCR و نژ نایبkohangen.com/wp-content/uploads/2019/01/Real-time-PCR...Real time PCR و نژ نایب:فیلات و نیودت)یژولونکتویب دشرا سانشرکا(