Human Cell Cycle PCR Array from SABiosciences Corporation

Several techniques have been developed for in vitro analysis of cell cycle, however, for in vivo studies, very few options are available especially when the samples are freshly frozen (FF) human tissues. In our lab, we have performed cell cycle gene expression in FF human liver biopsy tissues using the Human cell cycle RT Profiler PCR Array from SABiosciences. The RT Profiler PCR arrays have both the sensitivity of Real-time PCR and the profiling capability of microarray. These arrays are designed to analyze a panel of genes related to a disease state or biological pathway and are available for three different species that are human, mouse and rat.

The cell cycle PCR array is designed to analyze 84 genes that are crucial to cell cycle regulation, in a 96-well plate format. The 84 genes are involved in both positive and negative regulation of cell cycle, in the transitions between each of the phases, DNA replication and checkpoints of cell cycle. The plate also contains a panel of five housekeeping genes and a set of controls for genomic DNA contamination, RNA quality, and general PCR performance. As the expression level of housekeeping genes may vary among tissues or cells, the availability of more than one housekeeping gene provides convenience to the users to choose any of the uninfluenced genes for normalization. The procedure to complete the PCR array is very simple and starts by the conversion of experimental RNA into cDNA using first-strand synthesis. The synthesized cDNA is then mixed with ready-to-use PCR master mixes and subjected to PCR amplification in 96-well array plate that contain pre-dispensed gene specific primer sets. The relative expression is determined with a real-time PCR system and the delta-delta-Ct method.

In our lab, we have been working on impaired proliferation of hepatic cells in chronic hepatitis C virus (HCV) infection. Our study was aimed to identify the alterations in cell cycle gene expression with respect to early and advanced disease of chronic HCV infection. We analyzed the altered expression of cell cycle genes using the cell cycle PCR array and identified a list of differentially expressed genes involved at cell cycle checkpoints. We found cell cycle array very convenient to use as it saves time as well as the manual effort that could be required for the analysis of the same number of genes through conventional real-time PCR. Added to that, it is also sensitive and produces reproducible data. The presence of several housekeeping genes also helped us in selecting a single uninfluenced gene in our experiments, i.e GAPDH as normalization factor. However, the manufacturer recommends using only their reagent kits for isolation of RNA and cDNA synthesis to obtain completely reliable results. If the cost of these reagents is added to the cost of arrays then they are not very cost effective.