D N A M i cr o a rr a ys

D N A Microarrays

Sam Trammell

What is the need for Microarrays?

Northern blots for gene expression

B. WITEK-ZAWADA, A. KOJ. 2003. REGULATION OF EXPRESSION OF STROMYELYSIN-1 BY PROINFLAMMATORY CYTOKINES IN MOUSE BRAIN ASTROCYTES. Accessed from http://www.jpp.krakow.pl/journal/archive/1203/articles/02_article.html

Expanding our view of the cell

Northern blotting is limited in scope

Often we need to see expression levels of a wide range of genes on wide range of cell types

Provides a systematic manner to examine global gene expression patterns

Scale up to microarrays

Image from http://www.molecularstation.com/molecular-biology-images/508-microarray-pictures/68-affymetrix-microarray.html?size=big

Microarrays enable exploratory research

“A DNA microarray is an ordered array of nucleic acids, proteins, small molecules, that enables parallel analysis of complex biochemical samples.”

-Schena et al. (Science 270, 467-470, 1995)

Creating the Gene Chip

We must attach ssDNA or RNA oligonucleotides to a support surface

Covalent attachment strategies

Electrostatic adsorption strategies

Belosludtsev et al 2000. DNA microarray based on non-covalent oligonucleotide attachment and hybridization in two dimensions. Analytical Biochemistry

SpotBot

Customizable chips

Automated, large scale chip production

http://www.hhmi.org/biointeractive/media/gene_chips-lg.mov

Comparing Relative mRNA expression

Allows us to simultaneously analyze differential expression of genes between cell lines

Used to determine which genes are active and which are repressed in cancer cell lines

Fluorescent marking of mRNA occurs through reverse transcription

Image from http://cswww.essex.ac.uk/staff/W.Langdon/genechip/Image from http://cswww.essex.ac.uk/staff/W.Langdon/genechip/

Stringency of binding is controlled

Changing the salt/buffer concentration and the temperature alters the binding stringency

Low temperature/high Salt concentration yields low stringency

High stringency means only perfect matches anneal; lower stringency allows for some level of single

base differenceshttp://cswww.essex.ac.uk/staff/W.Langdon/genechip/

Hybridization of two lines to a chip

Reading the chip

Hybridization is quantized through fluorescent adsorption detected by a microarray chip scanner

http://www.moleculardevices.com/pages/instruments/gn_genepix4000.html

Statistical analysis of fluorescence

We must measure the ratio of fluorescence between the two dyes

Gain information of the relative level of expression compared to a standard cell

Comparing fluorescence levels

100 400

300 400

400 300

100 400

4 0.75

0.33 1

Green (normal) Fluorescence

Red (tumor) Fluorescence

Ratio Red to Green

A B

DC

Gene A Gene B Gene C Gene D

Red (tumor) Fluorescence

400 200 100 400

Green (Normal)Fluorescence

100 200 300 400

Ratio Red to Green

4 1 0.33 1


Sample 1 4 1 0.33 1

Sample 2 1 4 0.33 1

Sample 3 4 0.33 0.75 2

Sample 4 1 4 4 0.33

Sample 5 0.33 0.5 2 4

Sample 6 1 1 1 4

Sample 7 0.33 1 4 4

Compile ratios from many sample sets


Sample 1 0.650 0 0.333 -0.333

Sample 2 .6 -0.45 0 .650

Sample 3 0.100 0.500 -0.750 0.100

Sample 4 0 0.500 0.345 0.100

Sample 5 -0.550 -0.300 -0.335 -0.100

Sample 6 0.500 0.300 -0.100 0

Sample 7 0.300 0.110 -0.100 0.050

Transform each ratio by Log2

Assign each box a score based on this relative expression level

x10 x101:1

Repressed Induced


Sample 1 0.650 0 0.333 -0.333

Sample 2 .6 -0.45 0 .650

Sample 3 0.100 0.500 -0.750 0.100

Sample 4 0 0.500 0.345 0.100

Sample 5 -0.550 -0.300 -0.335 -0.100

Sample 6 0.500 0.300 -0.100 0

Sample 7 0.300 0.110 -0.100 0.050

Determining similarities in gene expression requires normalizing the data

To do this, we take the mean of each sample and divide by its standard deviation

We can then compare scores between each gene

Gene A Gene B Gene C

Gene A 1 0.5 -0.3

Gene B -0.5 1 0.3

Gene C 0.5 0.5 1

A positive score indicates similarity in expressionA score of zero indicates no similarity in expressionA negative score means expression is opposite (when one is induced, the other is repressed)

Hierarchical clustering is used to create a dendrogram

Gene A

Gene C

Gene B


Sample 1

0.650 0 0.333 -0.333

Sample 2

.6 -0.45 0 .650

Sample 3

0.100 0.500 -0.750 0.100

Sample 4

0 0.500 0.345 0.100

Sample 5

-0.550 -0.300 -0.335 -0.100

Sample 6

0.500 0.300 -0.100 0

Sample 7

0.300 0.110 -0.100 0.050


Sample 1

0.650 0 0.333 -0.333

Sample 2

.6 -0.45 0 .650

Sample 3

0.100 0.500 -0.750 0.100

Sample 4

0 0.500 0.345 0.100

Sample 5

-0.550 -0.300 -0.335 -0.100

Sample 6

0.500 0.300 -0.100 0

Sample 7

0.300 0.110 -0.100 0.050

Shyamsundar et al. 2005. A DNA microarray survey of gene expression in normal human tissues. Genome Biology.

Eisen et al. 1993. Cluster analysis and display of genome-wide expression patterns. PNAS.

Statistical programs exist to run through thousands of lines of data

Microarrays have diverse uses in research

Experimental uses of microarrays goes beyond comparative gene expression.

DNA Diagnostics and detectionSNP GenotypingMicroRNA profiling

ChIP on Chip analysis

A combined technique allowing researchers to examine transcriptional regulators and their control of gene expression

First a transcription factor is bound by an antibody and precipitated, then the annealed DNA strand is analyzed through a microrarray

Protein microarrays and antibody microarrays exist as well

Both detect protein expression

Antibody microarrays are essentially the same as ELISA

Image from http://newenglandbiolabs.de/en/index.php?option=com_content&task=view&id=70&Itemid=20

Tissue microarrays offer a clinical diagnostic tool

http://en.wikipedia.org/wiki/File:Rob7_melanoma.jpg

Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA

Microarray

Schena, M., Shalon, D., Davis, R., and Brown, P. (1995)

Main Method Used

What do you think it is?

?

Microarray!!!!!!!

Model Species UsedArabidopsis thaliana

Smallest genome of any higher eurkaryote

At time of paper, forty-five cloned Arabidopsis thaliana

Easy to store

Easy to obtain mutants

Taken from http://urgi.versailles.inra.fr/projects/GnpSNP/data_summary.php

Figure 1 and 2Color bars were callibratedwith signals from concentrations of human AChR mRNA.

Numbers and letters correspondto positions of each cDNA

Figure 1

Figure 2

Northern blot shows expression of CAB1, HAT4, ROC1, and human AChR

Schena et al.

Schena et al.

Table 1These are the positions of the significant genes hybridized in the study

a1,2 AChR Human AChR

b1,2 CABI Chlorophyll a/b binding

c11,12 rGR Rat Glucocortoid receptor

h11,12 TRP4 Yeast tryptophan biosynthesis

Schena et al.

Figure 1: A and B

The two scans differed in sensitivity

Result: Differential expression levels between the 45 genes tested

A: No hybridization between cDNA and mRNA for rat glucocorticoid receptor or yeast TRP4 targets at highest sensitivity

B: Allowed for a comparison

Schena et al.

Figure 1: C and D

One array scanned for either fluorescein-labeled cDNA (wt) or lissamine-labeled cDNA (HAT4-transgenic plant)

Intense expression of HAT4 in transgenic plant

50-fold elevation for HAT4

Schena et al.

Figure 1: E and F

Fluorescein-labeled cDNA from root tissue (E)

Lissamine-labeled cDNA from leaf tissue (F)

mRNA from light-regulated CAB1 gene was ~500 fold

26 other genes differed in expression by more than a factor of 5

Schena et al.

Figure 2No differential expression between wt and transgenic for CAB1

Expected differential expression between wt and transgenic for HAT4

No differential expression between wt and transgenic for ROC1

Schena et al.

Table 2: Comparison between microarrary and Northern blot

Schena et al.

Surprise!

HAT4 phenotype:elongated hypocotyls, early flowering, poor germination, altered pigmentation

Only 1 gene found with differential expression

Image retrieved from http://en.wikipedia.org/wiki/File:Salix_scouleriana.seed.jpg

Image retrieved from http://www.1up.com/do/my1Up?publicUserId=5685007

ConclusionsMicroarrays are really cool. Expressed Sequence Tags=EST 20,274 ESTs for A. thaliana

Uses?

Linking diseases and treatments with human gene sequences

Borevitz and Ecker. (2004)

I assume you can read that?

ESTs for A. thaliana: 190732

ESTs for H. sapiens: 5427257

Array Express websearch

Questions?

I recently read a paper that linked the composition of the human microbiome to obesity in twins. Along these lines, do you think that micro arrays could be used to assay the levels of gene expression of the human microbiome to study pathogens or other problems that may be due to microbes?

I recently read about a new type of microarray technique that is based on electrostatics. The article discussed how it is much more cost efficient, does not need complex chemical labels, and is more sensitive than traditional techniques. The article made it seem like this was going to revolutionize diagnostic and personalized medicine. I was just curious if you had come across anything else about this technique and whether it was in fact being used in labs or clinics?

Acknowledgements

Ahna Skop

University of Wisconsin-Madison

The attentive audience

Thank you for listening

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