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Methods and Instrumentation
GAIIx Technology (Illumina)
| Fig.1 Genome Analyzer IIx of the company Illumina |
For further information regarding the GAIIx system please go to the website of the company Illumina.
IScan Technology (Illumina)
| Fig.2 iScan Microarray Scanner of the company Illumina |
For further information regarding the iSCan system please go to the website of the company Illumina.
Agilent Technology
To perform high-dissolving scanning of microarrays the core facility genomics is using the “SureScan High-Resolution Technology” of the company Agilent (Fig. 3). For further information about the DNA Microarray Scanner please go to the website of the company Agilent Technologies.
| Fig. 3 Agilent Technologies DNA Microarray Scanner |
Applications
| Fig. 4 DNA Microarray Chip. Courtesy of Agilent Technologies. |
For further information regarding the DNA microarrays of the company Agilent please go to the website of the company Agilent Technologies Agilent Technologies.
Workflow of the Agilent Technologie
| Fig. 5 Workflow for sample preparation and array processing. Courtesy of Agilent Technologies. |
| Fig. 6 Schematic of amplified cRNA procedure. Generation of cRNA for a two-color microarray experiment is shown. When you generate targets for a one-color microarray experiment, only the Cy3-labeled “B” sample is produced and hybridized. Courtesy of Agilent Technologies. |
cDNA Microarray Technology
An organism exists by the complex and fine adjusted interaction of thousands of genes and their products. Traditional methods of molecular biology are based on the time-consuming and cost-intensive “ One gene in one experiment” approach. A comprehensive understanding of the complex interactions of the genome is only partly possible by this approach. Therefore a new technology, the DNA microarray, has been established by which great parts of the genome can be analysed within one experiment.
Microarrays are miniaturized sample carriers, on which thousands of known biomolecules can be located. The subsequent identification is based on the known position on the microarray. Thus microarrays represents highly miniaturized counterparts of the plates with the starting material (in this case by means of PCR amplified cDNA).
The aim of the DNA microarray analysis is a snapshot of the RNA pool of a tissue or a cell culture available at the moment of preparation. Therefore the definition “Transcriptome Analysis” is more precise than “Genomics”.
| Fig. 7 Basic procedure of assembling and assimilating cDNA- Microarrays (Diagramby Dr. Donauer; Image of the scanner by © Axon Instruments Inc.) |
In order to make a statement about the amount of a RNA to be analysed, the (“spotted”) DNA - the so called “Capture Probe” - which acts as binding partner has to be highly abundant. The before labelled cDNAs (symbolised by green and red dots in figure 7) obtained from the sample RNAs in the as hybridization defined reaction, can bind on the complementary DNA strands which are located on the array. The detection of the so immobilised cDNAs occurs on the bases of laser excitation of the bound fluorescence molecules.
chip. (Photo by M. Klein)
Processing of the cDNA libraries
The plasmid bearing bacteria are available as so called “cDNA libraries” in 96- or 384-well-plates. Based on these bacteria cultures are grown and capillary based plasmid preparations are performed. For that purpose a pipetting robot of the company Hamilton was especially adjusted to carry out the so called “minipreps” based on kits of the companies Eppendorf and Qiagen. Subsequently the cDNA is amplified by means of PCR and is distributed with the pipetting robot in 384 Well-plates. Before the printing the PCR products are dissolved in 3x SSC/1.5M Betain. Finally they are available in a concentration of about 50 ng/ml.
Printing of the Microarrays
The core of an arrayer is the printing head in which up to 32 split pins can be fixed. Based on a split in the pin the PCR product is migrating into the split by means of capillary force (fig. 12).
Up to 120 specially coated glass slides can be displayed on the arrayer. The printing head with the split pins moves from glass slide to glass slide and dispenses a small volume of the PCR product (volume about 1.2 nl, diameter about 100-150 micrometer).
Fig. 10 Our first arrayer, a self-construction of the year 2000 according to the instruction of the laboratory Pat Brown (stanford) (Photo by T. Sparna)
| Fig. 11 Commercial arrayer for a fully automatic production of micro- arrays according to the contact printing method.The instrument was built in 2004 by the company Genetix.(GB) and designed for a slide capacity of 96 pieces. (Photo by S. Teschner) |
After a thorough washing and drying stage the printing head takes new PCR products and distributes them similarly to the glass slides in direct closeness to the previous droplets (distance from point to point about 200 micrometer). That way we can presently locate more than 15.000 various PCR products on one single glass slide. Up to 120 identical microarrays are produced per print which presently requires 40 hours.
During the subsequent post-processing of the arrays the DNA molecules are fixed on the glass slide. In the exsiccator the microarrays have a durability of at least six weeks, however six months at the most.
| Fig. 12 Left: Microscopic picture of a capillary pin. The solution is absorbed into the capillary split and subsequently dispensed as droplets on the slide. Right: New printing head. |
Sample labelling and hybridization
The RNA (target probe) to be analysed is transcribed by a reverse transcription reaction into cDNA and at the same time labelled by means of fluorescence marked nucleotides (e.g. Cy3-dUPT). Parallel to it the same amount of reference RNA (s. under quality assurance) is labelled with another dye (e.g. Cy5-dUTP). After pooling both samples they are hybridized on the microarray over night (16h, 65°C). The target probes bind according to its amount at the capture probes on the array.
| Fig. 13 Labelling of the cDNA with different dyes takes place by an enzymatic reaction. (photo S. Teschner) |
Scanning of the hybridized arrays
After several steps of washing the hybridized microarrays are scanned with a two- color laser scanner (Axon 4000A). A false colour picture is generated which according to the international convention allocates Cy3 a green colour and Cy5 a red colour.
| Fig. 14 Evaluation of the hybridized microarrays by means of the program GenePix (Axon Instruments Inc.). Concerning the manual evaluation yet changes and flags in single spots are partly added. (Photo by M. Klein) |
Evaluation of the array data
For the analysis of the scanned microarrays a multitude of commercial and free accessible analysis soft wares is available. Based on these analysis tools artefacts are identified, the size of the dots is adjusted and for each dot a ratio is calculated which is a basis for further analyses.
| Fig. 15 "M" versus "A" Plot. (Photo by J. Wilpert) |