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REAL TIME PCR

USING SYBR GREENCourtesy of Dr. Hunt

University of South Carolina

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OVERVIEW

tissue

extract RNA

copy into cDNA(reverse transciptase)

do real-time PCR

analyze results

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REAL TIME PCR

• kinetic approach• early stages• while still linear

www.biorad.com

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SYBR GREEN CHEMISTRY

SS-DNA DS-DNA

5

0

200000000

400000000

600000000

800000000

1000000000

1200000000

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1600000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBERA

MO

UN

T O

F D

NA

110

1001000

10000100000

100000010000000

1000000001000000000

10000000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

CYCLE NUMBER AMOUNT OF DNA0 11 22 43 84 165 326 647 1288 2569 512

10 1,02411 2,04812 4,09613 8,19214 16,38415 32,76816 65,53617 131,07218 262,14419 524,28820 1,048,57621 2,097,15222 4,194,30423 8,388,60824 16,777,21625 33,554,43226 67,108,86427 134,217,72828 268,435,45629 536,870,91230 1,073,741,82431 1,400,000,00032 1,500,000,00033 1,550,000,00034 1,580,000,000

6

0

200000000

400000000

600000000

800000000

1000000000

1200000000

1400000000

1600000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

0

200000000

400000000

600000000

800000000

1000000000

1200000000

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1600000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AMOUNT OF DNA

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SERIES OF 10-FOLD DILUTIONS

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9

DDCt EFFICIENCY METHOD

APPROXIMATION METHOD

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IL1-b con

IL1-b vit

RPLP0 vit

RPLP0 con

1140

IL1-b con

IL1-b vit

RPLP0 vit

RPLP0 con

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IL1-b vit

RPLP0 vit

IL1-b con

RPLP0 con

av =19.80

av =19.93

av =18.03

av =29.63

D Ct = 9.70

D Ct = -1.7

D Ct = target - ref

D Ct = target - ref

Difference = DCt-DCt= DDCt = 9.70-(-1.7)= 11.40

control

experiment

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• 2 DDCt variant: assumes efficiency is 100% Fold change = 211.40 = 2702

• But our efficiency for IL1-beta is 93%– Fold change = 1.9311.40 = 1800

• Pfaffl equation corrected for RPLP0 efficiency – Fold change = 1901

DDCt = 11.40 for IL1-beta