Introduction
   Proteins are the macromolecules that carry out nearly all of the activities in the cells of living organisms, they are the molecular machines which make things happen. Therefore, the identification of the amounts and identity of proteins is necessary to understand the workings of a cellular systems. Proteins are indirectly produced from DNA (genes) through an intermediate molecule known as mRNA (Central Dogma of Biology). The transcription of genomic DNA to produce mRNA is the first process of protein synthesis, and differences in gene expression are responsible for both morphologic and phenotypic differences as well as indicative of cellular responses to environmental stimuli and perturbations.

   Measuring the amount and identity of large numbers of proteins can be difficult. Recent advances in the proteomics field are promising and in the near future this will be possible. However, identifying gene statement changes have traditionally been by measuring mRNA. The Northern blot places sample mRNA on a membrane, and a probe from a gene-of-interest in labeled and hybridized to the Northern blot. The amount of mRNA in the original sample is viewed as the amount of bound probe. A "dot-blot" has also been used for many years, but in this method the probe is bound to the membrane and the sample or "target mRNA" is labeled and hybridized. mRNA is extracted from sample cells or tissue, converted to complementary DNA (cDNA), amplified if necessary, and then labeled. The resulting labeled sample (targets) hybridizes with the DNA's from the known gene sequences (probes), that have been attached to a substrate.The identity and amount of labeled target bound yields information about theidentity and amount of mRNA expressed in the original sample.A "macroarray" is a "high-density" dot-blot where hundreds to thousands of probes are bound toa membrane. A "microarray" is a "very high density" dot-blot, where thousands to tens of thousands of probes are bound to a glass slide.

   To attach the probes to the surface of the substrates there are a number of techniques. A drop of each type of DNA in solution is placed onto a specially prepared glass microscope slide using a spotting tip. The DNA in the spots is bonded to the glass to keep it from washing off during the hybridization reaction, and the choice of DNA's to be used in the spots on a microarray determines which genes can be detected. By using a number of spotting tips in a spotting head and an arraying machine, a regular grid of thousands of spots in a small area can be produced rapidly.

   In order to detect cDNA's hybridized to the microarray, they must be labeled with a molecule that identifies their presence. In competitive hybridization green and red fluorescent dyes (fluors), such as rhodamine and fluorescein or Cy3 and Cy5 are commonly used for control and test cDNA. The actual fluors do not show their colors unless stimulated with a specific frequency of light by a laser, and the colors are not directly observed, but rather, a detector tuned to the wavelengths of the emitted light is used to measure the fluorescence. Once the cDNA has been labeled with the respective fluors, the hybridization can be done automatically in an automated slide processor, which is then scanned to produce the competitive gene expression results.
 
 

   By comparing the relative fluorescent intensities for equalized concentrations of the different cDNA's the relative up-regulation or down-regulation of certain genes in the test sample when compared to control can be determined. The spotting and hybridization of the DNA's is not always perfect, and careful image analysis must be performed to determine the accuracy of the results.

   The facilities we have here at SUNY Stony Brook include a genpakARRAY^TM 21 Robotic Micro-Arrayer System, and array scanner. Currently, with this system, we are capable of printing slides of up to 10,000 spots per slide and can generate 100 slides in 8 hours with accuracy to 1um. However, at this time we primarily do custom spotting of dozens to hundreds of probes per slide. The spotting facility is located in and sponsored by the Center for Biotechnology.

Affymetrix Gene chips
   The other facilities at Stony Brook include the GeneChip® Instrument System from Affymetrix. This system involves the use of fluorescently labeled single stranded DNA targets hybridizing to probe cells, about 50um by 50um in size, containing millions of copies of a specific oligonucleotide probe. Each gene chip contains about 50,000 different probes complementary to the genes of interest.

   To obtain the very sequence specific the oligonucleotide arrays, the gene chips are synthesized by photolithography, and the precise probe sequences are proprietary. Each gene is represented by twenty 25 mer oligos, grouped as 5’, middle, and 3’, and can be hybridized with total or polyA RNA targets.

   The Affymetrix systems represents a relatively turn-key and reproducible process. Hybridization results are determined both by perfect matching (PM) and mismatching (MM) so cross-hybridization contributes equally to both and cancels out, and quantitative RNA abundance can be determined by averaging pair wise differences of PM and MM.

   Affymetrix offers an extensive product line with gene chips for a number of organisms including human, rat, mouse, Arabidopsis, yeast and E.coli as well as a number of research applications including HIV, Cancer, Neurobiology, Toxicology, and Genotyping. The Affymetrix system at SUNY Stony Brook is located in the Center for Molecular Medicine and is sponsored by the School of Medicine.