Real-Time PCR I.

Pair the bands (2-6 on the gel) with the melting curves!

Real-Time PCR I.

Pair the bands (2-6 on the gel) with the melting curves!

2

3

4

5

6

Real-Time PCR II.

What can be said about the genotype of the 5 patient if the examined samples are STR sequences ?

2

3

4

5

6

Real-Time PCR II.

2 types of STR in each pearson. 2nd, 6th: homozygote, many repeat 3rds, 4th: heterozygote 5th: homozygote, few repeat

2

3

4

5

6

Real-Time PCR III. detection of point mutation

3 patients; „A” gene expression analysis

What is the conclusion?

What is the conclusion? 3 genotypes

Real-Time PCR gyakorlati alkalmazások I. pontmutáció detektálása

3 patients; „A” gene expression analysis

Real-Time PCR III. detection of point mutation Homozygote

wild type

What is the conclusion? 3 genotypes

3 patients; „A” gene expression analysis

Real-Time PCR III. detection of point mutation Homozygote

wild type

What is the conclusion? 3 genotypes

3 patients; „A” gene expression analysis

Heterozygote

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

What is the correlation between the disease and the rate of gene expression?

Healthy Diseased

Decreased mRNA copy number in diseased patient.

Healthy Diseased

Setting of the threshold cycle

Healthy Diseased

Healthy Diseased

Define Ct value!

Healthy Diseased

Healthy Diseased

Healthy: 21 Diseased: 31 ∆Ct=2 (Ctb-Cte)

Healthy Disease

DNA chip measuring gene expression

v

A B C D

0h 2h 4h 6h 8h 10h

What do you see?

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

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?

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

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

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

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

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

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

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