Table 2 (right) displays results obtained using 10 Human OneArray DNA Microarrays with 5 ug of total RNA from the Universal Reference RNA from Stratagene with one round of amplification using Amino Allyl MessageAmp™ Kit from Ambion.

For Hybridization, 10 ug of aRNA was labeled with Cyanine 5 fluorescent, and scanned with an Agilent scanner*.

*Agilent scanner data:
PMT: 100/10; Filter: F635.Median-B636.Mean>2.6B635sd

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GAM control probe is a Cy3 labeled 60-mer oligonucleotide probe designed from an alien sequence that does not cross-hybridize with human targets. More than 90 GAM features are evenly distributed across the arrayed area, and these features can be excited by a 532 nm laser without hybridization. GAM probes can serve as orientation or grid-alignment landmarks.

OGAM is a Cy3/Cy5 labeled 60-mer oligonucleotide probe designed from an alien sequence that does not cross-hybridize with human targets. It is located on top of the upper left corner of the print region and serves as an orientation landmark for template registration, as different scanners may have different image orientation output formats. Figure 2, below, illustrates this.

CGAMs are mixtures of Cy3/Cy5 labeled 60-mer oligo designed from an alien sequence that does not cross-hybridize with human targets. CGAMs are located at the four corners of the image and serve as the orientation landmarks for auto or manual grid alignment under Cy3 (532 nm) or Cy5 (635 nm) scanning channel. Figure 2, below, illustrates this.

IHCs contain mixtures of probes from 58 robustly expressed
house-keeping genes that serve as built-in positive controls. The probe mixture is dispensed at a fixed concentration, and more than 95 IHC features are evenly distributed across the arrayed area. The hybridization signal intensity of IHCs gives the indication of overall quality of sample processing and hybridization efficiency.

The IHL probes are dispensed in serial dilutions of IHC controls. The data extracted from IHL after target hybridization can be used to construct a hybridization intensity response curve depending on different dilutions of the probe mixture, which will be able to serve as an indicator to monitor the overall consistency of sample processing and hybridization stringency. Table 3, below, provides the ID number of the IHL controls and detailed information about their content.

Four probes per gene were selected from seven consistently expressed house-keeping genes. For each gene, one probe is designed from regions of 300-600 bp, 900-1200 bp, 1500-1800 bp, and 2100-2400 bp, respectively, from each 3’ end of the transcript. Tables 4 and 5, below, provide ITQC Probe information.

ETQC probes are alien probe sequences that do not cross-hybridize with human genomic probes on Human OneArray. The targets to these probes can be prepared separately as RNA containing a poly-A tail and spiked into the experimental RNA sample to monitor target preparation
quality and determine the sensitivity of the hybridization reaction.

Sequence information of Array Control RNA SPIKE #1~8 from Ambion, Inc., has been licensed for use with the Human OneArray microarray. Using the sequence information, eight 60-mer oligonucleotide probes are designed to compliment the respective RNA SPIKES. These probes are alien probes sequences that do not cross-hybridize with human genomic probes on the Human OneArray microarray, and thereby provide a set of of negative controls, which can be used as spike-in positive controls to determine the sensitivity of the hybridization process. Table 11, below, lists these probe ID numbers and their corresponding names.