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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

3quotesdbs_dbs28.pdfusesText_34