Microarray technology has been in use in the scientific community for the past few decades. This technique allows for obtaining data in an entire genome. Although enormous amounts of data can be obtained with a single experiment by this technique, excessive data can sometimes prove to be unmanageable. To overcome this problem, SA Biosciences has come up with the RT2
Profiler PCR array system. This system allows for analyzing the expression of a focused panel of genes using SYBR® Green based real-time PCR. This method combines the quantitative performance of real-time PCR with the multiple gene profiling of microarrays.
The Human Extracellular Matrix and Adhesion Molecules PCR Array is one of the arrays we have used successfully in our laboratory. This array profiles 84 genes involved in cell-cell and cell-matrix adhesion. These include adhesion molecules such as collagens, laminins and other basement membrane molecules. We were able to get meaningful data on the adhesion molecule gene expression using neural stem cells derived from fetal human brain. PCR arrays are available in both 96-well and 384-well formats; we used the 96-well plate format. The disadvantage with this format is that the primers for each gene are not replicated which may lead to erroneous results. However, the array does contain a panel of 5 housekeeping genes used for normalization of data, a panel of three reverse transcription controls, a genomic DNA control and three positive PCR controls along with 84 pathway based genes. The inclusion of several controls increases the validity of the assay. However, to get statistically meaningful data, assays with each condition need to be done at least in triplicate. This may become expensive if several pathways are to be tested. We had used the Bio-Rad iQ System for performing the assay using the 96-well plate formats. However, assay plates are available to suit several other instruments such as those from Life Technologies, Roche Applied Science, as well as from other companies.
The array comes as a set of four 96-well plates coated with primers for the genes of the pathway. Materials needed for RNA isolation, DNA decontamination, cDNA synthesis and PCR assay are to be supplied by the user. For this assay, high quality RNA isolation is essential. After RNA isolation, it is important to check the quality, quantity and integrity of the RNA; we do so using the Bioanalyzer from Agilent Technologies. Once good quality RNA is isolated, first-strand cDNA is synthesized and then used in the RT2 PCR assay.
The procedure for the assay itself is reasonably simple. Once the cDNA is prepared, it is mixed with your PCR reagents, and the mixture is then aliquoted into the provided 96-well primer-containing plates. The PCR reaction is then performed by thermal cycling in the real-time PCR instrument of choice. Once the data are collected, analysis is performed using the ddCt method. SA Biosciences offer free data analysis software on their website, which makes analysis very easy.
In summary, the Extracellular Matrix and Adhesion Molecules PCR Array from SA Biosciences is a very convenient tool for analyzing multiple genes involved in a single pathway simultaneously.