REAL TIME PCR SYBR GREEN
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1 REAL TIME PCR SYBR GREEN 1
2 THE PROBLEM NEED TO QUANTITATE DIFFERENCES IN mrna EXPRESSION SMALL AMOUNTS OF mrna LASER CAPTURE SMALL AMOUNTS OF TISSUE PRIMARY CELLS PRECIOUS REAGENTS 2
3 THE PROBLEM QUANTITATION OF mrna northern blotting ribonuclease protection assay insituhybridization PCR most sensitive can discriminate closely related mrnas technically simple but difficult to get truly quantitative results using conventional PCR 3
4 control NORTHERN expt target gene internal control gene actin, GAPDH, RPLP0 etc Corrected fold increase = 10/2 = 5 Ratio target gene in experimental/control = fold change in target gene fold change in reference gene 4
5 CYCLE NUMBER AMOUNT OF DNA , , , , , , , , , , ,048, ,097, ,194, ,388, ,777, ,554, ,108, ,217, ,435, ,870, ,073,741, ,400,000, ,500,000, ,550,000, ,580,000,000 5
6 CYCLE NUMBER AMOUNT OF DNA , , , , , , , , , , ,048, ,097, ,194, ,388, ,777, ,554, ,108, ,217, ,435, ,870, ,073,741, ,400,000, ,500,000, ,550,000, ,580,000,000 AMOUNT OF DNA AMOUNT OF DNA PCR CYCLE NUMBER PCR CYCLE NUMBER 6
7 AMOUNT OF DNA PCR CYCLE NUMBER 7
8 AMOUNT OF DNA PCR CYCLE NUMBER 8
9 Linear ~20 to ~1500 9
10 Linear ~20 to ~
11 REAL TIME PCR kinetic approach early stages while still linear 11
12 SERIES OF 10-FOLD DILUTIONS 12
13 13
14 14
15 threshold Ct SERIES OF 10-FOLD DILUTIONS 15
16 threshold =
17 threshold 17
18 STANDARD CURVE METHOD 18
19 dilutions target DNA triplicates cdna target primers dilutions reference DNA triplicates cdna reference primers Standard curve method 19
20 copy number target gene control copy number target gene experimental Dilution curve target gene NORTHERN control expt fold change in target gene= copy number experimental copy number control target gene internal control gene actin, GAPDH, RPLP0 etc Ratio experimental/control = fold change in target gene fold change in reference gene 20
21 copy number reference gene experimental Dilution curve reference gene copy number reference gene control NORTHERN control expt target gene internal control gene actin, GAPDH, RPLP0 etc Ratio experimental/control = fold change in target gene fold change in reference gene 21
22 PFAFFL METHOD M.W. Pfaffl, Nucleic Acids Research :
23 EFFECTS OF EFFICIENCY 23
24 CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA 100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY , ,048 1, ,096 2,213 1, ,192 4,205 2, ,384 7,990 3,748 1, ,768 15,181 6,747 2, ,536 28,844 12,144 4, ,072 54,804 21,859 8, , ,127 39,346 14, , ,842 70,824 23, ,048, , ,482 40, ,097, , ,468 69, ,194,304 1,356, , , ,388,608 2,578, , , ,777,216 4,898,763 1,338, , ,554,432 9,307,650 2,408, , ,108,864 17,684,534 4,335, , ,217,728 33,600,615 7,804,726 1,667, ,435,456 63,841,168 14,048,506 2,835, ,870, ,298,220 25,287,311 4,819, ,073,741, ,466,618 45,517,160 8,193,466 AFTER 1 CYCLE 100% = 2.00x 90% = 1.90x 80% = 1.80x 70% = 1.70x 24
25 CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA 100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY , ,048 1, ,096 2,213 1, ,192 4,205 2, ,384 7,990 3,748 1, ,768 15,181 6,747 2, ,536 28,844 12,144 4, ,072 54,804 21,859 8, , ,127 39,346 14, , ,842 70,824 23, ,048, , ,482 40, ,097, , ,468 69, ,194,304 1,356, , , ,388,608 2,578, , , ,777,216 4,898,763 1,338, , ,554,432 9,307,650 2,408, , ,108,864 17,684,534 4,335, , ,217,728 33,600,615 7,804,726 1,667, ,435,456 63,841,168 14,048,506 2,835, ,870, ,298,220 25,287,311 4,819, ,073,741, ,466,618 45,517,160 8,193,466 AFTER 1 CYCLE 100% = 2.00x 90% = 1.90x 80% = 1.80x 70% = 1.70x AFTER N CYCLES: fold increase = (efficiency) n 25
26 CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA 100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY , ,048 1, ,096 2,213 1, ,192 4,205 2, ,384 7,990 3,748 1, ,768 15,181 6,747 2, ,536 28,844 12,144 4, ,072 54,804 21,859 8, , ,127 39,346 14, , ,842 70,824 23, ,048, , ,482 40, ,097, , ,468 69, ,194,304 1,356, , , ,388,608 2,578, , , ,777,216 4,898,763 1,338, , ,554,432 9,307,650 2,408, , ,108,864 17,684,534 4,335, , ,217,728 33,600,615 7,804,726 1,667, ,435,456 63,841,168 14,048,506 2,835, ,870, ,298,220 25,287,311 4,819, ,073,741, ,466,618 45,517,160 8,193,466 AMOUNT OF DNA AMOUNT OF DNA 1,200,000, % EFF 1,000,000,000 90% EFF 80% EFF 800,000,000 70% EFF 600,000, ,000, ,000, PCR CYCLE NUMBER 10,000,000,000 1,000,000, % EFF 100,000,000 90% EFF 80% EFF 10,000,000 70% EFF 1,000, ,000 10,000 1, PCR CYCLE NUMBER
27 AMOUNT OF DNA 10,000,000,000 1,000,000, ,000,000 10,000,000 1,000, ,000 10,000 1, % EFF 90% EFF 80% EFF 70% EFF PCR CYCLE NUMBER 27
28 SERIES OF 10-FOLD DILUTIONS 28
29 threshold 29
30 QUALITY CONTROL -EFFICIENCY CURVES use pcr baseline subtraction (not curve fitting default option) set the threshold manually to lab standard check all melting curves are ok check slopes parallel in log view delete samples if multiple dilutions cross line together (usually at dilute end of curve) delete samples if can detect amplification at cycle 10 or earlier make sure there are 5 or more points check correlation coefficient more than
31 31
32 QUALITY CONTROL -EFFICIENCY CURVES use pcr baseline subtraction (not curve fitting default option) set the threshold manually to lab standard check all melting curves are ok check slopes parallel in log view delete samples if multiple dilutions cross line together (usually at dilute end of curve) delete samples if can detect amplification at cycle 10 or earlier make sure there are 5 or more points check correlation coefficient more than
33 PFAFFL METHOD M.W. Pfaffl, Nucleic Acids Research : NORTHERN target gene internal control gene actin, GAPDH, RPLP0 etc ratio = fold increase in target gene fold increase in reference gene 33
34 triplicates cdna triplicates cdna target primers reference primers 34
35 IL1-b vit RPLP0 con RPLP0 vit IL1-b con 35
36 IL1-beta IL1-b vit av =18.03 av =29.63 IL1-b con AFTER N CYCLES: increase = (efficiency) n AFTER N CYCLES: increase = (1.93) = = 2053 fold increase 36
37 RPLP0 RPLP0 con RPLP0 vit av =19.80 av =19.93 AFTER N CYCLES: increase = (efficiency) n AFTER N CYCLES: increase = (1.87) = = 1.08 fold increase 37
38 IL1-beta IL1-b vit RPLP0 RPLP0 con av =18.03 av =29.63 IL1-b con RPLP0 vit AFTER N CYCLES: increase = (efficiency) n AFTER N CYCLES: increase = (efficiency) n increase = (1.93) = = 2053 fold increase increase = (1.87) = = 1.08 fold increase ratio = change in IL1-B = 2053/1.08 = 1901 change in RPLP0 ratio = (E target ) Ct target (control-treated) (E ref ) Ct ref (control-treated) 38
39 Ct EFFICIENCY METHOD APPROXIMATION METHOD 39
40 IL1-b vit RPLP0 con RPLP0 vit IL1-b con 40
41 IL1-b vit RPLP0 con RPLP0 vit IL1-b con 41
42 control RPLP0 con Ct = target - ref Ct = 9.70 IL1-b con av =19.93 av =29.63 experiment IL1-b vit RPLP0 vit av =18.03 av =19.80 Ct = target - ref Ct = -1.7 Difference = Ct- Ct = Ct = 9.70-(-1.7) =
43 Ct = for IL1-beta If efficiency is 100% then Fold change = = 2702 Efficiency for IL1-beta is 93% Fold change = = 1800 Pfaffl equation corrected for RPLP0 efficiency Fold change =
44 SERIAL 10-FOLD DILUTIONS RED: 83% efficiency PURPLE: 93% efficiency 44
45 SERIAL 10-FOLD DILUTIONS RED: 94% efficiency PURPLE: 94% efficiency 45
46 Ct EFFICIENCY METHOD assumes minimal correction for the standard gene, or that standard and target have similar efficiencies 2 Ct variant assumes efficiencies are both 100% approximation method, but need to validate that assumptions are reasonably correct - do dilution curves to check Cts don t change The only extra information needed for the Pfaffl method is the reference gene efficiency, little more work than validating the approximation method 46
47 SPECIAL THANKS TO Dr. Joyce Nair-Menon and Lei Li for the use of their real-time PCR results Anyone who has ever discussed their realtime PCR results with me NEI - EY12711 for the money 47
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