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.

Slides:

Advertisements

Similar presentations

Real-Time quantitative PCR: Choices and Decisions

Advertisements

Polymerase Chain Reaction (PCR)

Overview of Real -Time PCR

REAL TIME PCR ………A step forward in medicine

Practical molecular biology

Fundamental in Real Time PCR

Tools for Molecular Biology Amplification. The PCR reaction is a way to quickly drive the exponential amplification of a small piece of DNA. PCR is a.

Analysis of gene expression by real-time PCR RBCS3 and Cab-1b transcript quantitation by real time PCR.

Real-Time PCR mRNA quantification. What do mRNA levels tell us? DNA  mRNA  protein Reflect level of gene expression Information about cell response.

PCR quantitative en temps réel Lydie Pradel. PCR.

PCR quantitativo.

Applied Biosystems 7900HT Fast Real-Time PCR System I. Real-time RT-PCR analysis of siRNA-induced knockdown in mammalian cells (Amit Berson, Mor Hanan.

An introduction to qPCR BCH452 Spring So far in BCH452 you have studied Carbohydrates: Glucose Proteins: ADH and LDH DNA: Plant DNA and viral DNA.

What Can You Do With qPCR?

Real Time PCR as a Diagnostic Tool. PCR = Polymerase Chain Reaction A way to make lots of copies of DNA What Happens in the Machine? Do the PCR virtual.

Q-PCR Bige Vardar

Real Time PCR = Quantitative PCR.

Real-Time PCR (Quantitative PCR)

Quantitative PCR Session 2: Overview of qPCR

COBAS AmpliPrep/Cobas TaqMan HIV-1 Test

Introduction to Real-Time PCR Joachim Hegstad Forsker Avdeling for Mikrobiologi og Smittevern Universitetssykehuset Nord-Norge Dept. for microbiology and.

Real time Pcr Dr. Basim Ayesh Biotechnology training course

Real-Time Quantitative RT-PCR

Analyzing your clone 1) FISH 2) “Restriction mapping” 3) Southern analysis : DNA 4) Northern analysis: RNA tells size tells which tissues or conditions.

DNA-based Methods for Quantifying Microbes in Atmospheric Samples Tom Hill, Helen Ahern and Bruce Moffett University of East London.

Quantitative Real-Time PCR Adrien Six Sophie Dulauroy Institut Pasteur & Université Pierre et Marie.

IN THE NAME OF GOD Alireza Azizi Alireza Salehi Mohsen Rahmanian.

Dr. Sumbul Fatma Department of Medical Biochemistry.

Quantitative Real Time PCR USING SYBR GREEN. SYBR Green SYBR Green is a cyanine dye that binds to double stranded DNA. When it is bound to D.S. DNA it.

Real time RT-PCR Quantitating Gene Expression.

Quantification of RNA by real-time PCR

Results Alien Reference RNA QRT-PCR Detection Kit for Monitoring the Overall Performance of QRT-PCR Assays Bahram Arezi, Melissa McCarthy & Holly Hogrefe.

PCR reaction to be visualized “in real time” as the reaction progresses to quantify the amount of DNA in the sample at the start of the reaction REAL TIME.

How do you identify and clone a gene of interest? Shotgun approach? Is there a better way?

qPCR SNAPSHOTS LIVE MAY 13, 2003.

Real-Time Quantitative PCR Basis

HCV PCR By Henrietta Orji July 31 st, 2010 Hepatitis C Virus by Polymerase Chain Reaction.

Figure 1: Basic Principle Of PCR * Poor precision * Low sensitivity * Short dynamic range < 2 logs * Low resolution * Non-automated * Size-based discrimination.

The Application of Real-Time PCR in the Diagnosis of Infectious Disease The Application of Real-Time PCR in the Diagnosis of Infectious Disease T.P.Sloots.

Molecular Testing and Clinical Diagnosis

1. 2 VARIANTS OF PCR APPLICATIONS OF PCR MECHANICS OF PCR WHAT IS PCR? PRIMER DESIGN.

Taqman Technology and Its Application to Epidemiology Yuko You, M.S., Ph.D. EPI 243, May 15 th, 2008.

Polymerase Chain Reaction (PCR) Nahla Bakhamis. Multiple copies of specific DNA sequences; ‘Molecular Photocopying’

PCR With PCR it is possible to amplify a single piece of DNA, or a very small number of pieces of DNA, over many cycles, generating millions of copies.

R EAL TIME P CR 1. L IMITATIONS OF E ND -P OINT PCR Poor Precision Low sensitivity Low resolution Non - Automated Size-based discrimination only Results.

SYBR Green I Chemistry Detects all double stranded DNA

Introduction to quantitative real- time PCR Veryan Codd

PCR is amplification of DNA in a tube What to put in the PCR tube?? Template DNA DNA cDNA obtained by reverse transcription of mRNA Or Cell free.

Good qPCR The Necessary and the Reasonable

Performance of Applied Biosystems™ consumables on Bio-Rad CFX™ Real-Time PCR Detection System February 15, 2017.

Application of Rapid-Cycle Real-Time Polymerase Chain Reaction for the Detection of Microbial Pathogens: The Mayo-Roche Rapid Anthrax Test James R. Uhl,

Principles of Real-Time Quantitative PCR Techniques

Gel electrophoresis analysis Automated DNA analyzer.

Nucleic acid-based methods (I)

Israel maritime college

RT-PCR ANALYSIS NOHA L. IBRAHIM.

Minimal Residual Disease (MRD) Detection Technologies

Application of Rapid-Cycle Real-Time Polymerase Chain Reaction for the Detection of Microbial Pathogens: The Mayo-Roche Rapid Anthrax Test James R. Uhl,

DNA Technology.

Rapid detection of resistance in Mycobacterium tuberculosis: a review discussing molecular approaches D. García de Viedma Clinical Microbiology and.

Gene quantification using real-time quantitative PCR

Nucleic acid-based methods (I)

Development and validation of a real-time quantification assay to detect and monitor BRAFV600E mutations in hairy cell leukemia by Susanne Schnittger,

Applied Biosystems™ Consumables on Bio-Rad CFX™

Real-time PCR in the microbiology laboratory

Presentation transcript:

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 480 Applied Biosystems 7500 Fast BiomarkOpenArray Real time PCR Instruments

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

Roche Lightcycler

Real time PCR: an overview

One or two step RT-PCR?

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

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