Oligo GEArray® Focused DNA Microarray:
A robust, flexible and cost effective tool of gene expression profiling

2. Performance of the Oligo GEArray:

With the combination of a 3D nylon matrix, an improved RNA linear amplification and labeling method and chemiluminescent detection, the Oligo GEArray® system delivers performance comparable to other microarrays. To characterize system performance more closely, a series of hybridization experiments has been performed with known quantities of labeled synthetic RNA target spiked into a labeled human RNA sample. The Oligo GEArray detects the target in the 10 fM range (Figure 1) at a spiking ratio of 1:20,000,000. The signal for the synthetic target also displays a linear response over three orders of magnitude range of concentration. The low limit of detection and wide linear response range allows the Oligo GEArray to detect low abundance transcripts.

Manufacture of the Oligo GEArrays utilizes an advanced non-contact inkjet type printing process that ensures good array printing quality and reproducibility. Figure 2 displays a representative comparison between two hybridization replicates. The typical median percent Coefficient of Variation is routinely less than 10. Table 2 summarizes the performance of the Oligo GEArray.

Figure 2: The Oligo GEArray® exhibits excellent array-to-array reproducibility. XpressRef™ Human Universal Reference Total RNA (GA-004) was converted to labeled cRNA target using the TrueLabeling-AMP™ Linear RNA Amplification Kit (GA-010). Equal amounts (2 µg) of amplified product was hybridized to separate Human Oligo GEArray® Trial Microarrays. The Chemiluminescent Detection Kit (D-01) was used to detect the presence of labeled target on the arrays. The raw intensity values for each gene from one array were plotted against those values from the second array, and the data were fit to a straight line having a correlation factor (R2) of 0.9939. The result demonstrates that raw Oligo GEArray® data is very reproducible from one array to the next.

3. Target Amplification & Labeling:

In vitro transcription based RNA amplification methods have been used for many microarray platforms. The method provides excellent amplification power while preserving the original transcript profile. Amplification allows the use the smaller amounts of input RNA and the detection of lower abundance messages. However, the current methods are both time-consuming and costly. SABiosciences has designed an easier and more cost effective method for the Oligo GEArray, the TrueLabeling-Amp Linear RNA Amplification Kit. The protocol requires only one tube reaction and only one purification step and occurs in less than six hours. This method delivers expression profiles nearly identical to those generated using the traditional, longer, more tedious and more expensive procedures (Figure 3). The TrueLabeling-Amp Linear RNA Amplification Kit is an integral part of the Oligo GEArray system providing it with excellent amplification performance. Table 3 summarizes the characteristics of the TrueLabeling-Amp Linear RNA Amplification Kit.

4. Array Detection Options:

The Chemiluminescent Detection Kit from SABiosciences already provides sensitive detection for both its Oligo GEArrays and cDNA GEArrays. The GEArray microarray system requires no special equipment and offers flexible detection options. The chemiluminescent detection method permits the use of either highly sensitive cooled CCD camera based imagers or basic X-ray film to record array images. Methodology is now available for the single-color fluorescent detection of the GEArray. Briefly, streptavidin conjugated to the Cy5 fluorophore replaces the alkaline phosphatase conjugate normally used for chemiluminescence. The GEArray is then dried and mounted on a glass slide. The slide-mounted GEArray can be analyzed on any commercial fluorescent scanner. The fluorescent and chemiluminescent detection methods yield nearly identical gene expression profiles (Figure 4).

Figure 4. The Fluorescent and Chemiluminescent Detection Methods give similar GEArray results. XpressRef™ Human Universal Reference Total RNA (GA-004) was converted to labeled cRNA target using the TrueLabeling-AMP™ Linear RNA Amplification Kit (GA-010). Equal amounts of amplified product was hybridized to separate Oligo GEArray® Human Hematology Microarrays (OHS-801). Either the Chemiluminescent Detection Kit (D-01) (Right Image, acquired with an Alpha Innotech FluorChem 8800 CCD camera) of the fluorescent method of detection (Left Image, scanned on an Axon 4000B instrument) was used to detect the presence of labeled target on the arrays. The result indicates that both detection methods yield similar gene expression profiles.

Summary:

The Oligo GEArray represents each gene in its application- and pathway-specific gene lists with a carefully designed gene-specific oligonucleotide. The use of oligos, as opposed to gene-specific cDNA fragments for example, permits faster development of microarray content and potentially increases the specificity of target hybridization and the resulting array signal. Characteristics common between the Oligo and cDNA GEArray include the high-binding capacity of the nylon membrane and the Chemiluminescent Detection Method. When also combined with the new TrueLabeling-AMP Linear RNA Amplification Kit, all of these properties help provide the Oligo GEArray systems with sensitivity, wide dynamic ranges, and reproducibility comparable to all other microarray platforms. The TrueLabeling-AMP method also imparts to the Oligo GEArray the ability to detect lower abundance messages using smaller amounts of input RNA in a time and cost effective manner. The increased convenience of the labeling method does not compromise its ability to provide an equally accurate representation of RNA samples as the traditional methods. Finally, the development of a fluorescent method of detection increases the flexibility of the Oligo GEArray platform. In these ways, the Oligo GEArray further advances the state of microarray technology.