Microarrays have become a popular tool for studying gene expression in a single experiment, offering higher throughput capabilities than other methods such as Northern blot or RT-PCR. The Superarray Bioscience GEArray Q series provides a simple method for the characterization of gene expression specific to biological pathways or disease states in a comprehensive and cost effective manner. It is also essay to run, sensitive and applicable for routine use in a variety of research laboratories. Most comprehensive commercial array products produce a huge amount of data that can be overwhelming and difficult to interpret. In addition, they are typically expensive and require special equipment for experimentation (eg. hybridization station and specialized scanners). For the GEArray Q no special equipment is needed apart from basic insruments such as a thermal cycler for probe synthesis, hybridization oven for Array hybridization and washing and CCD camera or X-ray film for image acquisition.
The GEArray Q contains up to 96 cDNA sequences from genes associated with a specific biological pathway. Gene specific cDNA fragments are printed on a 3.8 x 4.8 cm nylon membrane with an advanced non-contact printing technology. Each sequence fragment is printed in a tetra-spot format, which provides a signal in an easily identifiable pattern. The membrane is spotted with a negative control (pUC18 DNA), blank and housekeeping genes (including GAPDH, cyclophilin A and ribosomal protein L13a). Each kit contains multiple arrays that allow researchers to run a parallel and comprehensive study of multiple samples or different conditions however, it is designed for one-time use only. Finally, SupeArray offers software for one month for data analysis with no cost to the user.
The technique requires from 1-5 ug of isolated total RNA for probe generation. Checking the absorbance reading of isolated RNA is recommended, not only to determine the concentration, but also to assess its purity from proteins and free nucleotides. RNA isolation is followed by synthesis of the cDNA probe using biotin labelled 16-dUTP, which I highly recommend over the radioactive dUTP. During probe generation, the array membrane is pre-hybridized with GEAprehyb for 2 h, followed by hybridization overnight at 60C with continuous agitation (in the presence of the biotin labelled cDNA probe). Hybridization solution, which contains the labelled cDNA probe, can be kept at 4C to be used for another experiment. After hybridization, the membrane is washed with different stringencies of SSC washing solutions, followed by soaking in the blocking solution. The membrane is incubated with chemiluminescent substrate for 2 min. I recommend suing CDP-Star for chemiluminescent detection because it reaches peak light emission between 2-4 h, allowing time to determine the highest signal with lowest background. Finally, the image is acquired either by CCD or X-ray film.
The initial results (or the real raw data) from a GEArray experiment is a greyscale digital image of the array spots. Generation of the image involves either an integrated imager, such as a CCD based system, or a digitized image of developed X-ray film using a flatbed scanner. The digitized image is then analyzed using GEArray Expression Analysis Software. The relative level of gene expression is directly related to the spot intensity. In our laboratory, we used the GEArray Q Series (Mouse Extracellular Matrix and Adhesion Molecules Gene Array), to investigate genes which may play a key role on atherosclerosis development. Using these arrays, we successfully identified three genes which need further confirmation. Generating knockout model animals for these genes will be the final goal.
Nasser Al-Shanti, Ph.D.
Research Fellow
IRM
Manchester Metropolitan University