ABI PCR ABI 7000 Cq Rn Sample Threshold

ABI 荧光定量PCR仪 ABI 7000

阈值和Cq值 Rn + Sample Threshold Cq Baseline No Template Rn -

PCR理论方程 Where: n Rn=Total fluorescence signal n RB=Base fluorescence signal n XO=Initial number of target molecules n EX=Efficiency of target amplification n n =Number of cycle n RS=Fluorescence signal per target molecule

当n=Cq时，Rn=Rq，即设定的阈值 Rq =RB+XO(1+Ex)Cq. Rs log(Rq -RB)=log XO +Cqlog(1+ Ex)+log. Rs Cqlog(1+ Ex)=log(Rq -RB)-log XO -log. Rs Cq=a·log XO +b

化学原理 SYBR® Green I Taq. Man®探针

SYBR® Green I 熔解曲线

转基因大豆检测 %GMO=A/B （A, EPSPS; B, lectin) log(%GMO)=log(A/B)=log. A-log. B=ΔC(q)*M DNA from Standard/Sample TUBE 1 PCR mix Lectin primers TUBE 2 PCR mix GMO primers

Standards/ Sample C(q) epsps C(q) Lectin ∆C(q) 0 ND 22. 6 ND 0. 1 ND 22. 6 ND 0. 5 28. 2 22. 5 5. 7 1. 0 27. 2 22. 5 4. 7 2. 0 26. 2 22. 8 3. 4 5. 0 24. 6 22. 7 1. 9 Soy Dessert 24. 6 22. 4 2. 1 Soy Flour ND 22. 2 ND Soy Burger 24. 2 23. 4 0. 7 Log percent GMO 转基因大豆检测结果 y = -0. 264 x + 1. 202 R 2= 0. 998 0. 80 0. 60 0. 40 0. 20 0. 00 -0. 20 -0. 40 0 1 Standards/ Sample 2 3 ∆ C(q) 4 5 6 ∆C(q) %GMO Soy Dessert 2. 1 4. 40 Soy Flour ND < 0. 5 Soy Burger 0. 7 >5

双标准曲线 Color 1 – FAM detection for ERBB 2 Color 2 - VIC detection for GAPDH

样品扩增：正常vs肿瘤 PMT 1 -FAM detection- ERBB 2 肿瘤 PMT 2 -VIC detection- GAPDH 肿瘤 正常 正常

实验结果 ERBB 2表达 Multiplex Reactions: C(q) Healthy RNA Carcinoma 28. 40 27. 36 28. 46 27. 12 28. 43± 0. 04 26. 24 ± 0. 17 GAPDH表达 Multiplex Reactions: C(q) Healthy RNA Carcinoma 23. 27 22. 81 23. 41 22. 86 23. 34± 0. 10 22. 83 ± 0. 03

实验结果 ERBB 2 GAPDH Healthy RNA Tumor RNA 1095 1052 95500 85730 2130 2492 Relative Expression 136000 130800 ERBB 2 copies / GAPDH copies Healthy RNA 1095 / 95500 = 0. 011 1052 / 85730 = 0. 012 0. 011 Tumor RNA 2130/136000 = 0. 017 2492/130800 = 0. 019 0. 018 2 1. 8 1. 6 1. 4 1. 2 1 0. 8 0. 6 0. 4 0. 2 0 Healthy Tissue Carc. Tissue 0. 018/ 0. 011＝ 1. 63

2 -ΔΔc(t) 法 ΔΔC(t) = 4. 41 -5. 09 =-0. 68± 0. 18 2 -ΔΔc(t) =1. 41~1. 82 Healthy RNA ERBB 2 GAPDH ΔC(t) 28. 40 23. 27 5. 13 28. 46 23. 41 5. 05 28. 43± 0. 04 23. 34± 0. 10 5. 09± 0. 18 结果与双标准曲线法相近 Carcinoma RNA ERBB 2 GAPDH ΔC(t) 27. 36 22. 81 4. 55 27. 12 22. 86 4. 26 26. 24 ± 0. 17 22. 83 ± 0. 03 4. 41 ± 0. 21

SNP检测 单核苷酸多态性 Single Nucleotide Polymorphisms AACCTGCATAATGCCAG AACCTGCA GAATGCCAG