Microarrays and Transcript Profiling

Gene expression patterns are traditionally studied using Northern blots (DNA-RNA hybridization assays). This approach involves separation of total or polyA+ RNA on agarose gels and after transfer to a nitrocellulose membranes probing with radioactively labeled DNA corresponding to gene/sequence of interest. Hybridization of the labeled DNA with mRNA is recognized via radioactive detection (i.e. autoradiography). The band position provides information about message size and its intensity provides an estimate of the level of expression. Analysis of various samples collected at different time points or tissue/organs provides information on the developmental regulation of expression.

Main problems with Northern analysis: Slow and laborious to perform Not applicable to high throughput genomics scale analysis

Microarrays Microarrays are physically ordered sets of DNA molecules Each DNA molecule is placed onto the array at a precisely defined location Allow assay of thousands of genes/sequences simultaneously, quickly and efficiently

Microarrays analysis was developed in response to the availability of large and expanding databases of anonymous sequences (mostly cDNA and ESTs). Microarray analysis provides information on the genes pattern of expression as a function of time, physiological treatment, the tissue topology and histology in a massively-parallel manner.

Two basic types of microarrays

cDNA Microarrays Frequently composed of EST library clones Transcription profiling (Expression analysis)

Oligonucleotide arrays Also called “DNA Chips” 10-mer sequences synthesized in situ

Recent technology allow production of microarrays composed of 70-mers (essentially a hybrid of the two techniques)

cDNA Microarrays Robotically spoted cDNAs in the form of an arrayed grid onto a microscope slide. The spots are spaced at microscopic (200-500 micron) intervals and have an initial volume of about 1-5 nl.

Method of spotting

Precision positioning robot, carrying printing quill(s)

Probes and the printing process

cDNA Microarrays • A DNA microarray is a solid surface containing a number of different DNA molecules • These molecules (termed “probes”) are individually printed onto the solid surface, using

robotic techniques • The individual array elements are circles, having diameters of about 100 µm, and spaced at

center-to-center distances of about 180 µm • This means a standard glass slide can contain as many as 25,00 different elements

slides (100)slides (100)

print headprint head

microtiter platesmicrotiter plates

• The microarray probes are produced by PCR amplification either of different inserts from cDNA libraries or genomic DNA

• High throughput methods are needed for the amplification and purification procedures, to ensure quality control and to keep track of different amplicons

• Alternatively, synthetic oligonucleotide sequences can be deposited on the surface

Oligonucleotide arrays (DNA Chips) • A DNA chip is a solid surface, on which are immobilized a large number of different DNA

molecules • The molecules are produced by step wise synthesis in situ • Two companies have competing methods:

Affymetrix using photolithographic techniques Agilent using modified ink-jet deposition technologies

Microarray analysis • DNA microarrays and DNA chips are analyzed by hybridization, using fluorescent

“targets” produced from mRNA extracted from the tissue, at a time point and condition of interest.

• The mRNA molecules are typically extracted pair-wise from two different tissues, corresponding to the treatment and the control.

• The mRNAs are separately converted into fluorescent molecules by reverse-transcription in the presence of two different fluorochromes (Cy3 and Cy5).

• The targets are mixed and hybridized to the arrays. The intensities of the hybridization signals are then measured.

• The hybridiztion is done at high stringency, and the slide is then scanned at the two fluorescent emission wavelengths to measure the fluorescence intensities in the different spots à The high sensitivity results from small hybridization volume, so that you can employ

high target concentration. This exceeds the concentration typically used in Northern blots by about 105. This means you can potentially detect nearly all cellular transcripts.

à The high specificity results from use of long hybridization targets, and stringent hybridization and wash conditions. No detectable cross-hybridization is observed between closely related members of gene families (>70% nucleotide identity over long regions).

• Microarrays therefore provide a means to globally analyze the concentrations of different RNA within cells.

Arabidopsis thaliana microarray: >18,000 EST elements Considerations when working with microarrays: Choice of format Microarray printing Microarray hybridization Microarray scanning Data analysis and archiving Extract RNA from cells under first condition Prepare cDNA in the presence of Cy3 dUTP Extract RNA from cells under second condition (control) Prepare cDNA in the presence of Cy5 dUTP Mix fluorescent cDNAs, hybridize to arrays and scan image.

Cy5 image

Cy3 image

Cy5 image

Cy3 image

Microarray analysis

• The intensity values for the two channels (Cy3 and Cy5) are determined and the values exported to spreadsheets.

• Massively-parllel global analysis of gene expression changes becomes possible. à In statistics, traditional paradigm is a few variables and many replicates. With

microarrays, you have many variables and few replicates. Analysis of massively-parallel information is very difficult to address.

• Clustering techniques can be used to infer functions of unknown genes, and to define hierarchical patterns of gene regulation.

Analyzing microarray data • Microarray based results need to be data-based and made widely available

• Standardization- how to construct a fingerprint record again which to compare new array data

• Need for community-wide standards and controls

• Need to create a structured “controlled” vocabulary for microarray data

• Need for open systems architecture

A schematic representation of six components of a microarray experiment (MIAME Standards)..

Minimum information about a microarray experiment (MIAME)

Brazma et al. 2001. Minimum information about a microarray experiment (MIAME)—toward standards for microarray data. Nature Genetics 29: 365-371