real-time pcr i. -...
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![Page 1: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/1.jpg)
![Page 2: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/2.jpg)
Real-Time PCR I.
Pair the bands (2-6 on the gel) with the melting curves!
![Page 3: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/3.jpg)
Real-Time PCR I.
Pair the bands (2-6 on the gel) with the melting curves!
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![Page 4: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/4.jpg)
Real-Time PCR II.
What can be said about the genotype of the 5 patient if the examined samples are STR sequences ?
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![Page 5: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/5.jpg)
Real-Time PCR II.
2 types of STR in each pearson. 2nd, 6th: homozygote, many repeat 3rds, 4th: heterozygote 5th: homozygote, few repeat
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![Page 6: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/6.jpg)
Real-Time PCR III. detection of point mutation
3 patients; „A” gene expression analysis
What is the conclusion?
![Page 7: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/7.jpg)
What is the conclusion? 3 genotypes
Real-Time PCR gyakorlati alkalmazások I. pontmutáció detektálása
3 patients; „A” gene expression analysis
![Page 8: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/8.jpg)
Real-Time PCR III. detection of point mutation Homozygote
wild type
What is the conclusion? 3 genotypes
3 patients; „A” gene expression analysis
![Page 9: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/9.jpg)
Real-Time PCR III. detection of point mutation Homozygote
wild type
What is the conclusion? 3 genotypes
3 patients; „A” gene expression analysis
Heterozygote
![Page 10: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/10.jpg)
Real-Time PCR III. detection of point mutation Homozygote
wild type
What is the conclusion? 3 genotypes
3 patients; „A” gene expression analysis
Heterozygote
Homozygote mutant
![Page 11: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/11.jpg)
What is the correlation between the disease and the rate of gene expression?
Healthy Diseased
![Page 12: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/12.jpg)
Decreased mRNA copy number in diseased patient.
Healthy Diseased
![Page 13: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/13.jpg)
Setting of the threshold cycle
Healthy Diseased
![Page 14: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/14.jpg)
Healthy Diseased
![Page 15: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/15.jpg)
Define Ct value!
Healthy Diseased
![Page 16: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/16.jpg)
Healthy Diseased
![Page 17: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/17.jpg)
Healthy: 21 Diseased: 31 ∆Ct=2 (Ctb-Cte)
Healthy Disease
![Page 18: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/18.jpg)
DNA chip measuring gene expression
v
A B C D
0h 2h 4h 6h 8h 10h
What do you see?
![Page 19: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/19.jpg)
v
A B C D
0h 2h 4h 6h 8h 10h
A gene continous, steady state expression (house keeping gene), B gene decreases, C increases, D no expression
DNA chip measuring gene expression
![Page 20: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/20.jpg)
Measuring similarity between expression patterns
Gene name
0h 2h 4h 6h 8h 12h
Q 1 2 3 4 3 2
S 1 3 4 4 3 2
T 1 2 3 4 3 2
V 4 6 8 6 4 2
Q and T genes similar because of the same ratios at each time point. How similar it its response to that of genes S and Q?
![Page 21: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/21.jpg)
Gene name
0h 2h 4h 6h 8h 12h
Q 1 2 3 4 3 2
S 1 3 4 4 3 2
T 1 2 3 4 3 2
V 4 6 8 6 4 2
Pearson-correlation coefficient It quantifies the extent to which the expression patterns of two genes go up together and down together over several time points.
Measuring similarity between expression patterns
![Page 22: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/22.jpg)
Gene name
0h 2h 4h 6h 8h 12h
Q 1 2 3 4 3 2
S 1 3 4 4 3 2
T 1 2 3 4 3 2
V 4 6 8 6 4 2
Pearson-correlation coefficient It quantifies the extent to which the expression patterns of two genes go up together and down together over several time points. =1: expression patterns of the 2 genes track perfectly =-1: expression patterns of the 2 genes track perfectly, but in opposition to the another. 0: expression patterns of the two genes do not track each other at all.
Measuring similarity between expression patterns
![Page 23: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/23.jpg)
Gene name
0h 2h 4h 6h 8h 12h
Q 1 2 3 4 3 2
S 1 3 4 4 3 2
T 1 2 3 4 3 2
V 4 6 8 6 4 2
Find the correlation between Q and S genes: 1: compute the sample mean and sample standard deviation of the expression values of each genes. Xs=2,83 SS=1,067 Xq= 2,5 Sq= 0,957
Measuring similarity between expression patterns
![Page 24: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/24.jpg)
Gene name
0h 2h 4h 6h 8h 12h
Q 1 2 3 4 3 2
S 1 3 4 4 3 2
T 1 2 3 4 3 2
V 4 6 8 6 4 2
Find the correlation between Q and S genes: 2: substract Xs from each value in the S row and divide each result by Ss. Do the same in the Q row, to produce the following normalized row. Snorm: -1,7; 0,16; 1,1; 1,1; 0,16; -0,5 Qnorm: -1,5; -0,5; 0,5; 1,5; 0,5; -0,5
Measuring similarity between expression patterns
![Page 25: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/25.jpg)
Gene name
0h 2h 4h 6h 8h 12h
Q 1 2 3 4 3 2
S 1 3 4 4 3 2
T 1 2 3 4 3 2
V 4 6 8 6 4 2
Find the correlation between Q and S genes: Multiply the first number in Snorm by the first number in Q norm, the second number in Snorm….. And so on, keeping a running sum of these products. Divide this sum by the number of elements in each row (6) to get the correlation coefficient.
Measuring similarity between expression patterns
![Page 26: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/26.jpg)
Gene name
0h 2h 4h 6h 8h 12h
Q 1 2 3 4 3 2
S 1 3 4 4 3 2
T 1 2 3 4 3 2
V 4 6 8 6 4 2
Find the correlation between Q and S genes: ρ(Q,S)=0,897
Measuring similarity between expression patterns
![Page 27: Real-Time PCR I. - web.med.u-szeged.huweb.med.u-szeged.hu/mdbio/eng/materials/2011-2012/2nd_semester/cell_p/...Real-Time PCR I. Pair the bands (2-6 on the gel) with the melting curves!](https://reader030.vdocuments.mx/reader030/viewer/2022041206/5d5c171788c993b6038b520e/html5/thumbnails/27.jpg)
Gene name
0h 2h 4h 6h 8h 12h
Q 1 2 3 4 3 2
S 1 3 4 4 3 2
T 1 2 3 4 3 2
V 4 6 8 6 4 2
Home work: find the correlation numbe r between T and V genes Ρ (T,V)= -1
Measuring similarity between expression patterns