Human OneArray^® Controls

Orientation Settings and Controls Dye Intensity Controls Positive Controls Negative Controls

Below is a brief description of the Human OneArray experimental control features and instructions of how to extract this information for analysis. SimpleMeasure is a free, small, Java-based applet designed to automatically extract this data and generate graphs similar to those below. The program can be downloaded here.

Orientation Settings and Controls

Orientation Grid Alignment Mark (OGAM)   Probe ID Number: PH_c_0000089   Purpose: To define the orientation of the scanned image   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.   Corner Grid Alignment Marks (CGAM)   Probe ID Number: PH_c_0000072   Purpose: To define the corners of the scanned image, and to assist with image analysis.   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 1: Human OneArray OGAM and CGAM Positions

Figure 2: Human OneArray GAM Positions Grid Alignment Mark (GAM)   Probe ID Number: PH_c_0000001   Purpose: To assist in adjusting the scanner settings; to monitor the scanner performance across the scanned region.   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.   Analysis Instructions:   Convert scanned data into Excel format Sort data by column ‘ID’ Copy ~90 rows of data with ID PH_c_0000001 Obtain the median, mean, and standard deviation of the signals in the ‘F532 Median’ and ‘F532 Mean’ columns.   Expected Outcome: When the suggested scanner settings from the OneArray User Guide are used, saturated or near-saturated signal should be observed for all GAM probes.

Dye Intensity Controls

Cy3 and Cy5 Intensity Ladder   Probe ID Numbers: Cy3 Intensity Ladder PH_c_0000002 to PH_c_0000009 Cy5 Intensity Ladder PH_c_0000010 to PH_c_0000017 Purpose: To assist in adjusting the scanner settings. Cy3 and Cy5 Intensity Ladder probes are Cy3- or Cy5-labeled 60-mer oligonucleotide probes designed from an alien sequence that do not cross-hybridize with human targets. Eight dilutions, and three to six replicates of each dilution can be excited by a 532 nm(Cy3) or 635 nm (Cy5) laser without hybridization. Analysis Instructions: Convert scanned data into Excel format Sort data by column name ‘ID’ Copy ~90 rows of data with IDs above. Obtain the mean of the 3-6 replicates signals in the ‘F532 Median’ and ‘F532 Mean’ columns. Expected Outcome: When the suggested scanner settings from the OneArray User Guide are used, saturated or near-saturated signal should be observed for probes PH_c_0000008 and PH_c_0000016. Scanner settings should be adjusted so that the signal increases linearly from the lowest concentration unit in order to observe the saturation level. Cy3 Cy5 Probe Concentration Unit PH_c_0000002 PH_c_0000010 0.39 PH_c_0000003 PH_c_0000011 0.78 PH_c_0000004 PH_c_0000012 1.56 PH_c_0000005 PH_c_0000013 3.125 PH_c_0000006 PH_c_0000014 6.25 PH_c_0000007 PH_c_0000015 12.50 PH_c_0000008 PH_c_0000016 25.00 PH_c_0000009 PH_c_0000017 50.00 Table 1: Human OneArray Cy3 and Cy5 Ladder Probe information ID F635 Median F635 Mean PH_c_0000010 10608 9956 PH_c_0000010 11833 11321 PH_c_0000010 11059 10475 PH_c_0000010 12748 11764 PH_c_0000010 11106 9708 PH_c_0000010 7991 7105 PH_c_0000011 19303 18580 PH_c_0000011 24135 23471 PH_c_0000011 19090 18596 PH_c_0000011 24472 23160 PH_c_0000011 24490 21312 PH_c_0000011 19917 17913 PH_c_0000012 37772 32030 PH_c_0000012 48964 43739 PH_c_0000012 51195 45869 PH_c_0000012 33185 29470 PH_c_0000012 31362 29326 PH_c_0000012 39620 34003 PH_c_0000013 65535 49856 PH_c_0000013 65535 47857 PH_c_0000013 65535 50867 PH_c_0000013 65535 51355 PH_c_0000013 65535 52266 PH_c_0000013 45684 40202 PH_c_0000014 65535 58240 PH_c_0000014 65535 54896 PH_c_0000014 65535 55110 PH_c_0000014 65535 54016 PH_c_0000014 65535 58050 PH_c_0000014 65535 58093 PH_c_0000015 65535 60572 PH_c_0000015 65535 62503 PH_c_0000015 65535 59844 PH_c_0000015 65535 58715 PH_c_0000015 65535 59599 PH_c_0000015 65535 62979 PH_c_0000016 65535 62595 PH_c_0000016 65535 59930 PH_c_0000016 65535 58309 PH_c_0000016 65535 59135 PH_c_0000016 65535 60074 PH_c_0000016 65535 60463 PH_c_0000017 65535 63049 PH_c_0000017 65535 61676 PH_c_0000017 65535 63046 PH_c_0000017 65535 59504 PH_c_0000017 65535 59036 PH_c_0000017 65535 63695 Table 2: Sample Cy5 Ladder Control Probe ID F635 Median F635 Mean PH_c_0000010 10891 10055 PH_c_0000011 21901 20505 PH_c_0000012 40350 35740 PH_c_0000013 62227 48734 PH_c_0000014 65535 56401 PH_c_0000015 65535 60702 PH_c_0000016 65535 60084 PH_c_0000017 65535 61668 Table 3: Sample Cy5 Ladder Control Output Figure 3: Sample Cy5 Ladder Control Output

Positive Controls

Intrinsic Hybridization Controls (IHCs)   Probe ID Number: PH_c_0000025 Purpose: To monitor the overall quality of sample processing and hybridization efficiency. IHCs contain a mixture of probes from 58 robustly expressed housekeeping 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. NOTE: Occasionally, some spots will be missing or misaligned, so the intensity results can be removed for a more accurate average. Figure 4: IHC + IHL Output Intrinsic Hybridization Ladder (IHL)   Probe ID Numbers: PH_c_0000026 to PH_c_0000033 Purpose: To monitor the overall quality of sample processing and hybridization efficiency. The IHL probes are dispensed in serial dilutions of IHCs. 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 serves as an indicator of the overall consistency of sample processing and hybridization stringency. Analysis Instructions Convert scanned data into Excel format. Sort data by column name ‘ID’. Copy rows of data with IDs above. Obtain the mean of the 3-6 replicate signals in the ‘F532 Median’ and ‘F635 Median’ columns. Expected Outcome: When the suggested scanner settings from the OneArray User Guide are used, saturated or near-saturated signal should be observed for all IHC probes. Scanner settings should be adjusted so that the signal increases linearly from the lowest to highest concentration IHL.

Intrinsic Target Quality Control (ITQC)   Probe ID Numbers: PH_c_0000038 to PH_c_0000065 Purpose: To monitor the length of the targets for assessment of the quality of sample integrity and processing. Four probes per gene were selected from seven robustly expressed housekeeping genes. For each gene, one probe is designed from regions of 300-600 bp, 900-1200 bp, 1500-1800 bp, and 2100-2400 bp from the 3’ end of each transcript. Analysis Instructions: Convert scanned data into Excel format. Sort data by column name ‘ID’. Copy rows of data with IDs PH_c_0000038 to PH_c_0000065. Obtain the mean of the 3-6 replicates signals in the ‘F532 Median’ and ‘F635 Median’ column. Expected Outcome: When high quality targets are used, signals from all the probes can be observed. It is common to observe higher signals from probes designed closer to the 3’ end of the transcript. ITQC Gene Number Gene Name C_ITQC2 Lysosomal-associated membrane protein 1 C_ITQC3 Calnexin C_ITQC4 Ubiquitin specific protease 11 C_ITQC5 Damage-specific DNA binding protein 1 C_ITQC6 Ubiquitin-activating enzyme 28 C_ITQC7 Tripartite motif-containing 28 C_ITQC8 Actin Binding LIM protein 1 Table 4: ITQC Genes ITQC Gene No. 300-600 nt 900-1200 nt 1500-1800 nt 2100-2400 nt C_ITQC2 PH_c_0000038 PH_c_0000039 PH_c_0000040 PH_c_0000041 C_ITQC3 PH_c_0000042 PH_c_0000043 PH_c_0000044 PH_c_0000045 C_ITQC4 PH_c_0000046 PH_c_0000047 PH_c_0000048 PH_c_0000049 C_ITQC5 PH_c_0000050 PH_c_0000051 PH_c_0000052 PH_c_0000053 C_ITQC6 PH_c_0000054 PH_c_0000055 PH_c_0000056 PH_c_0000057 C_ITQC7 PH_c_0000058 PH_c_0000059 PH_c_0000060 PH_c_0000061 C_ITQC8 PH_c_0000062 PH_c_0000063 PH_c_0000064 PH_c_0000065 Table 5: ITQC Transcript Locations Probe ID F635 Median F532 Median PH_c_0000038 3,554 4,339 PH_c_0000039 1,303 1,059 PH_c_0000040 1,548 768 PH_c_0000041 840 431       PH_c_0000042 27,979 21,191 PH_c_0000043 9,053 6,186 PH_c_0000044 4,154 3,104 PH_c_0000045 4,192 2,699       PH_c_0000046 3,365 7,855 PH_c_0000047 1,742 7,780 PH_c_0000048 1,076 4,601 PH_c_0000049 907 2,894       PH_c_0000050 3,885 2,775 PH_c_0000051 4,076 2,523 PH_c_0000052 1,953 1,046 PH_c_0000053 355 191 Probe ID F635 Median F532 Median PH_c_0000054 5,661 5,099 PH_c_0000055 2,201 1,477 PH_c_0000056 660 459 PH_c_0000057 642 429       PH_c_0000058 6,241 3,409 PH_c_0000059 1,000 653 PH_c_0000060 714 280 PH_c_0000061 244 121       PH_c_0000062 3,132 23,108 PH_c_0000063 774 4,676 PH_c_0000064 1,041 6,347 PH_c_0000065 168 752 Table 6: Averaged ITQC Intensities Figure 5: ITQC Output

Negative Controls

Ambion ArrayControl™ RNA SPIKE #1-8   Probe ID Numbers: AM_c_0000015 to AM_c_0000022 Purpose: To monitor the specificity of hybridization; can be used with spike-in positive controls to determine the sensitivity of the hybridization and troubleshoot target preparation and labeling reactions. Sequence information for ArrayControl™ RNA SPIKE #1-8 from Ambion, Inc., has been licensed for use with Human OneArray microarrays. Using the sequence information, eight 60-mer oligonucleotide probes are designed to complement the respective RNA SPIKES. These probes are alien probe sequences that do not cross-hybridize with human genomic probes on the Human OneArray DNA microarray. Analysis Instructions: Convert scanned data into Excel format Sort data by column name ‘ID’ Copy rows of data with IDs AM_c_0000015 to AM_c_0000022 Obtain the mean of the 3-6 replicates signals in the ‘F532 Median’ and ‘F635 Median’ columns. Expected Outcome: To learn more about the usage of RNA SPIKES, please visit Applied Biosystems Ambion website: http://www.ambion.com. Probe ID # Ambion ArrayControl™ RNA SPIKE ID # AM_c_0000015 Array Control RNA SPIKE 1 AM_c_0000016 Array Control RNA SPIKE 2 AM_c_0000017 Array Control RNA SPIKE 3 AM_c_0000018 Array Control RNA SPIKE 4 AM_c_0000019 Array Control RNA SPIKE 5 AM_c_0000020 Array Control RNA SPIKE 6 AM_c_0000021 Array Control RNA SPIKE 7 AM_c_0000022 Array Control RNA SPIKE 8 Table 7: Ambion ArrayControl™ RNA SPIKES Probe ID # F635 Mean F532 Mean AM_c_0000015 79 72 AM_c_0000016 211 75 AM_c_0000017 139 97 AM_c_0000018 137 86 AM_c_0000019 213 90 AM_c_0000020 142 82 AM_c_0000021 103 95 AM_c_0000022 66 66 Table 8: Ambion ArrayControl™ RNA SPIKES Sample Averaged Intensities Figure 6: Ambion ArrayControl™ RNA SPIKES Sample Output

Extrinsic Target Quality Control (ETQC)   Probe ID Numbers: PH_c_0000066 to PH_c_0000071 Purpose: To monitor the specificity of hybridization. ETQC probes are alien probe sequences that do not cross-hybridize with human genomic probes on Human OneArray DNA microarray. The targets to these probes can be prepared separately as RNA containing poly-A tail and spiked into the experimental RNA sample to monitor target preparation quality and determine the sensitivity of the hybridization reaction. Analysis Instructions: Convert scanned data into Excel format Sort data by column name ‘ID’ Copy rows of data with IDs PH_c_0000066 to PH_c_0000071 Obtain the mean of the 3-6 replicates signals in the ‘F532 Median’ and ‘F635 Median’ columns. Expected Outcome: These probes emit background-level signal. Targets matching to these probes can be obtained and used in a manner similar to the Ambion ArrayControl™ RNA SPIKE controls. Probe ID Database Reference PH_c_0000066 NM_202341.1 (Entrez Gene ID: 842522) PH_c_0000067 NM_122927.2 (Entrez Gene ID: 833497) PH_c_0000068 NM_001036207.1 (Entrez Gene ID: 843854) PH_c_0000069 NM_104167.4 (Entrez Gene ID: 841722) PH_c_0000070 NM_129890.3 (Entrez Gene ID: 818930) PH_c_0000071 NM_106286.3 (Entrez Gene ID: 843969) Table 9: ETQC Control Reference Probe ID F635 Median F532 Median PH_c_0000066 50 74 PH_c_0000067 59 79 PH_c_0000068 73 89 PH_c_0000069 49 84 PH_c_0000070 87 98 PH_c_0000071 46 79 Table 10: ETQC Sample Data Figure 7: ETQC Sample Output