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1 REAL TIME PCR USING SYBR GREEN Courtesy of Dr. Hunt University of South Carolina
2 OVERVIEW tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR analyze results
3 REAL TIME PCR kinetic approach early stages while still linear
4 SYBR GREEN CHEMISTRY SS-DNA DS-DNA
7 SERIES OF 10-FOLD DILUTIONS
9 Ct EFFICIENCY METHOD APPROXIMATION METHOD
10 IL1-b con IL1-b vit RPLP0 vit RPLP0 con
12 IL1-b vit RPLP0 vit IL1-b con RPLP0 con av =19.80 av =19.93 av =18.03 av =29.63 Ct = 9.70 Ct = -1.7 Ct = target - ref Difference = Ct- Ct = Ct = 9.70-(-1.7) = control experiment
13 2 Ct variant: assumes efficiency is 100% Fold change = = Ct variant: assumes efficiency is 100% Fold change = = 2702 But our efficiency for IL1-beta is 93% But our efficiency for IL1-beta is 93% –Fold change = = 1800 Pfaffl equation corrected for RPLP0 efficiency Pfaffl equation corrected for RPLP0 efficiency –Fold change = 1901 Ct = for IL1-beta Ct = for IL1-beta
1 REAL TIME PCR USING SYBR GREEN. 2 THE PROBLEM NEED TO QUANTITATE DIFFERENCES IN mRNA EXPRESSION SMALL AMOUNTS OF mRNA –LASER CAPTURE –SMALL AMOUNTS.
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