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ICoBioSE Real-Time QPCR Application Workshop - Gene Expression Analysis

This document provides an overview of real-time quantitative PCR (qPCR). It defines real-time qPCR as a PCR-based technique that couples DNA amplification with quantification. The document discusses applications like gene expression analysis and disease diagnosis. It also covers experimental design considerations for real-time qPCR like choices of one-step vs two-step reverse transcription and relative vs absolute quantification methods. Detection methods for real-time qPCR like SYBR-based and probe-based techniques are also outlined.

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afifatul achyar
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98 views

ICoBioSE Real-Time QPCR Application Workshop - Gene Expression Analysis

This document provides an overview of real-time quantitative PCR (qPCR). It defines real-time qPCR as a PCR-based technique that couples DNA amplification with quantification. The document discusses applications like gene expression analysis and disease diagnosis. It also covers experimental design considerations for real-time qPCR like choices of one-step vs two-step reverse transcription and relative vs absolute quantification methods. Detection methods for real-time qPCR like SYBR-based and probe-based techniques are also outlined.

Uploaded by

afifatul achyar
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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I C o B i o S E

Real-Time qPCR Application Workshop


GENE EXPRESSION ANALYSIS
AFIFATUL ACHYAR, M.Si.
CONTENTS
What is real-time PCR?

Application of real-time PCR

Choices of Experimental Design


Quantitative PCR (qPCR), also called real-time PCR or quantitative
What is
real-time PCR, is a PCR-based technique that couples amplification
real-time of a target DNA sequence with quantification of the concentration
quantitative of that DNA species in the reaction.
PCR?

PCR real-time PCR


• PCR is a process for the amplification of Real-time PCR is a specialized technique that
specific fragment of DNA allows a PCR reaction to be visualized “in real
• Visualized using gel agarose electrophoresis real-time quantitative PCR time” as the reaction progress
APPLICATIONS OF REAL-TIME qPCR

Gene Expression Analysis Disease Diagnosis & Management


• Cancer research • Viral quantification
• Drug research • Bacterial quantification

Food Testing Animal and Plant


• GMO (Genetically Modified Breeding
Organism) food
• Gene copy number
• Porcine gene detection
(Halal Testing)
CHOICES OF EXPERIMENTAL DESIGN If your sample is RNA Template,
then you have to choose:
One-Step Reverse Transcription (RT) qPCR
Reverse transcription & real time PCR simultaneously

What is your sample? RNA Template 5 Two-Step Reverse Transcription (RT) qPCR
• Gene expression analysis
• Viral load of RNA virus
4 6 • Reverse transcription: RNA to cDNA
• Real time PCR

DNA Template Relative quantification PCR


• Bacterial quantification
• Single Nucleotide Polymorphisms
3 7 • Amplification of target gene
• Amplification of Quantification
• GMO/Halal Testing reference/housekeeping gene methods
CHOICES • Expression result in RATIO
Probe-Based
Sequence-specific
2 8 Absolute quantification PCR
• Need of standard to generate standard curve
Detections of PCR • Result: copy number gene
product in real-time SYBR-based
• ds-DNA intercalating dye 1 9 The choice is depend on your research
• binding to ds-DNA & budget
• Melting curve analysis
DETECTION OF PCR PRODUCTS IN REAL TIME_FLUOROPHORE

SYBR Green Probe-based: TaqMan Probes Probe-based: FRET Probes


• ds-DNA intercalating dye
• Sequence-specific • Sequence-specific
• binding to ds-DNA
• Quantification at elongation step • Quantification at annealing step
• Specific target sequence primer
• Melting curve analysis
REVERSE TRANSCRIPTION (RT) PROCEDURES

One-Step RT PCR

Two-Step RT PCR
QUANTIFICATION METHODS

RELATIVE QUANTIFICATION real time PCR ABSOLUTE QUANTIFICATION real time PCR
• Amplification of target gene • Need of standard to generate standard curve
• Amplification of reference/housekeeping gene • Standard: This is a sample of known concentration or copy
• Referece gene: whose expression level does not change under the number used to construct a standard curve
experimental conditions or between different tissues • Type of standards:
• Expression result in RATIO 1. RNA standard
• Methods of calculation: 2. DNA standard:
1. Livak Methods: CT methods • plasmid DNA (cloning of target gene)
2. The ∆CT Method Using a Reference Gene • PCR fragment
3. The Pfaffl Method --> when amplification efficiency target = • Genomic DNA
reference • Result: copy number gene OR concentration

Threshold cycle (CT) or crossing point (Cp) or cycle of quantification (Cq): The cycle at which the amplification plot crosses
the threshold (i.e., there is a significant detectable increase in fluorescence).
Video

Polymerase Chain Reactions

https://www.youtube.com/watch?v=MyLrs_h1OlE

Overview of qPCR

https://www.youtube.com/watch?v=1kvy17ugI4w

Real time PCR Data Analysis Tutorial

https://www.youtube.com/watch?v=GQOnX1-SUrI
THANK YOU

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