1 real time pcr using sybr green courtesy of dr. hunt university of south carolina
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1
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
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0
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1000000000
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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
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0
200000000
400000000
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800000000
1000000000
1200000000
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0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
AM
OU
NT
OF
DN
A
0
200000000
400000000
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800000000
1000000000
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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
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