1. 1 REAL TIME PCR USING SYBR GREEN Courtesy of Dr. Hunt University of South Carolina

2. 2 OVERVIEW tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR analyze results

3. 3 REAL TIME PCR kinetic approach early stages while still linear www.biorad.com

4. 4 SYBR GREEN CHEMISTRY SS-DNA DS-DNA

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7. 7 SERIES OF 10-FOLD DILUTIONS

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9. 9 Ct EFFICIENCY METHOD APPROXIMATION METHOD

10. 10 IL1-b con IL1-b vit RPLP0 vit RPLP0 con

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12. 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) = 11.40 control experiment

13. 13 2 Ct variant: assumes efficiency is 100% Fold change = 2 11.40 = 2702 2 Ct variant: assumes efficiency is 100% Fold change = 2 11.40 = 2702 But our efficiency for IL1-beta is 93% But our efficiency for IL1-beta is 93% –Fold change = 1.93 11.40 = 1800 Pfaffl equation corrected for RPLP0 efficiency Pfaffl equation corrected for RPLP0 efficiency –Fold change = 1901 Ct = 11.40 for IL1-beta Ct = 11.40 for IL1-beta