Real-time reverse-transcription PCR is the most sensitive and reliable method for gene expression analysis. However, using a conventional approach, real-time PCR assays can only monitor a few genes at time, making the analysis of many genes all at once very tedious and time-consuming. Microarray-based profiling technologies allow the expression of many genes (almost the whole genome) to be profiled in one experiment by including multiple gene-specific nucleic acid probes on a solid support; each probe represents a specific gene. However, variability in the results has been considered a major drawback. Thus, technology which might feature the profiling capability of microarray and the quantitative nature of real-time PCR is desired. SuperArray Bioscience Corporation has released RT2
Profiler™ PCR Array technology. The PCR Array takes advantage of real-time PCR performance and combines it with the ability of microarrays to detect the expression of panels of genes simultaneously.
Each RT2 Profiler™ PCR Array is a 96-well plate containing gene-specific primer sets for a thoroughly researched set of 84 relevant, pathway-or disease-focused genes plus five housekeeping genes and two negative controls. These primer sets are optimized for use with RT2 Profiler™ PCR master mixes for SYBR Green real-time detection, providing the PCR Arrays with superior sensitivity, with linear dynamic ranges, and the flexibility to be used on most real-time instrumentation. The flexibility and simplicity of the PCR Arrays make them accessible for routine use in every research laboratory.
In our laboratory, we have used RT2 Profiler™ PCR Arrays for gene expression profiling in human cancer cell lines. Since the only step involved is the addition of cDNA with SYBR Green PCR master mixture, the PCR Arrays are performed within less than half of the time of a conventional real-time PCR assay. The assay is sensitive and reproducible both within and between assays. This is probably due to the elimination of errors associated with multiple steps.
However, the price of the array has to be taken into account. Unlike microarrays where one can profile almost the whole genome, with the RT2 Profiler™ PCR Array, one can profile only 84 genes. Thus, completely unbiased profiling is not possible. Additionally, 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 the PCR arrays and labor, the RT2 Profiler™ PCR Arrays do not provide any benefit over microarrays. Additionally, there are too many control wells, which may be unnecessary and the wells might be better utilized for gene expression purposes. We believe 5 wells/sample may be sufficient for housekeeping genes and other controls. It is also important to mention that the manufacturer custom designs the arrays with a minimum order of 24 plates (cost/array approx the same as pre-designed plates). Thus, we strongly recommend that the custom designed arrays be used as an extremely sensitive, reproducible, practical and convenient way to obtain gene expression profile of a set of selected genes (for example, 11 genes) for a small but significant number of important samples (for example, 6 samples). In using the RT2 Profiler™ PCR Arrays for such a purpose, the cost may be more than justified.
RT2 Profiler™ PCR Array From SuperArray Biosciences
The RT<sup>2</sup> Profiler™ PCR Array takes advantage of real-time PCR performance and combines it with the ability of microarrays to detect the expression of panels of genes simultaneously.
Expensive for routine use if the entire plate is used for gene expression profiling of one sample at a time.
The Bottom Line
If properly designed, the arrays may be an extremely sensitive, reproducible, practical and convenient way to obtain a gene expression profile of a set of selected genes for a small, but significant number of important samples. In using RT<sup>2</sup> Profiler™ PCRArrays for such a purpose, the cost may be more than justified.