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Methods for qPCR Analysis

23 avr. 2003 qPCR Analysis. Renée Horner. Queen of qPCR. Ambion Inc. rhorner@ambion.com ... Methods of Analysis. •Absolute quantitation.



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What does qPCR measure? If you are measuring gene expression qPCR will tell you how much of a specific Definitions of the terms found in the analysis.



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Searches related to qpcr analysis PDF

The key equipment for qPCR is a specialized thermocycler with fluorescence detection modules which is used to monitor and record the fluorescence in real-time as amplification occurs A typical workflow of qPCR for gene expression measurement involves RNA isolation reverse transcription qPCR assay development qPCR experiment and data analysis

What is qPCR data analysis?

Gene expression analysis by real-time qPCR has been a key enabler of a routine and robust approach for measuring gene expression in genes of interest, as well as monitoring biomarkers. This section will provide the key features of qPCR data analysis and describe examples of common methods to analyze data from a qPCR assay.

What is the exponential phase of qPCR?

Important Parameters of Quantitative PCR (qPCR) Analysis Exponential Phase It is important to quantitate your qPCR at the early part of the exponential phase of amplification instead at the later cycles or at the plateau. At the beginning of the exponential phase, all reagents are still in excess.

How does a qPCR machine measure fluorescence?

The qPCR machine measures the intensity of fluorescence emitted by the probe at each cycle. During the first cycles, there is not enough fluorescence to be detected, but the reaction rapidly produces more and more amplicons and the fluorescence builds up. A qPCR curve has typically an exponential phase followed by a plateau phase.

What is a typical workflow of qPCR for gene expression measurement?

typical workflow of qPCR for gene expression measurement involves RNA isolation, reverse transcription, qPCR assay development, qPCR experiment and data analysis. Special attention is needed for preventing RNA degradation.

Methods for qPCR Analysis

Methods for

qPCR Analysis

Renée Horner

Queen of qPCR

Ambion, Inc.

rhorner@ambion.com Date:

Wed, 23 Apr 2003

From: "Dr Stephen A Bustin" To: "Renee Horner"

Subject:

Re: UK NA quantification meeting

Methods of Analysis

Absolute quantitation

Relative quantitation

Comparative quantitation

Why absolute

quantitation?

Gives a measure of copy number

Viral load determination

FDA filing

Inter-lab comparisons

Why is absolute quantitation

not currently feasible?

There is no reliable method for

preparing, quantitating and storing RNA standards

No NIST traceable standards

Next Best

Alternatives?

Synthetic templates known to

come up at a certain Ct value-"semi quantitative PCR"

Why relative

quantitation?

Does not require that you know the

copy numbers for the standard curve

Can be used to determine fold

increases and decreases in gene expression

There is no need to "over optimize"

the efficiencies

What is needed for

relative quantitation?

Any sample that can be used as a

comparison for other samples-"calibrator"

A serial dilution of the calibrator to

give a standard curve in terms of 1x, 2x, 10x, etc

Relative qPCR Data

GOI

E= 43%

Normalizer

E= 68%

qPCR Gene

Expression Analysis

S a m p l e GOI N o r m G OI N o r m T r e a t e d U n t r e a t e d U n t r eat e d 1 2 5. 01 45.
9 9 0 5 4 1 0 0 T r eat ed 1 1 6. 05 14. 2 6 1 1 3 2 0 7 U n t r eat e d 2 35.
4 0 8 9. 10 0. 40
1. 00 T r eat ed 2 42.
7 5 5 7. 72
0. 74
1. 86

In both animals, the GOI is

expressed twice as much as in the treated areas as the untreated areas. This data verifies the array data.

Why comparative

quantitation?

Mathematical determination of

relative quantities

No standard curve needed

Higher throughput

Best used when particular ratios

are expected or are verifying a "trend"

What is needed for

comparative quantitation?

Calibrator sample used as a 1x standard

Samples that are prepared identically

Ideally, if normalizing the results, your

GOI and the normalizer

will have the same efficiency

Comparative Quantitation

Ct GOI Ct norm Ct Ct

Sample

Ct

Calibrator

Ct

Relative quantity = 2

Ct

Genotypin

g

Experimental Rationale

Genome Equivalents norm

Normalized Equivalents

Genome Equivalents GOI

Sample Type

Homozygous

2 2 1.0

Heterozygous

1 2 0.5 Null 2 00 0 qPCR Genotype Analysis W e l l D y e R ep l i cat e C t E 1 F A M b 22.
26
F 1 F A M b 22.
29
E 1 HE X b 26.
05 F 1 HE X b 26.
03 A3 F A M c 4 0 A4 F A M c 4 0 A 3 HE X c 24.
84
A 4 HE X c 24.
17 A 7 F A M s 19. 52
A 8 F A M s 19. 1 A 7 HE X s 23.
92
A 8 HE X s 22.
33
H11 F A M z p 40
H12 F A M z p 40
H11 H E X z p 24.
88
H12 H E X z p 26.
04 wt CalibratorSample MC3 0 5

Sample AS10

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