OneArray® Mouse Microarray v2 Controls

Orientation Settings and Controls

Dye Intensity Controls

Positive Controls

Negative Controls		 Below is a brief description of the Mouse OneArray experimental control features and instructions of how to extract this information for analysis.


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 Mouse 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 mouse 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.
OGAM_CGAM position

Figure 1: Mouse OneArray OGAM and CGAM Positions
GAM.gif

Figure 2: Mouse 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 mouse 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:
 
  1. Convert scanned data into Excel format
  2. Sort data by column 'ID'
  3. Copy ~90 rows of data with ID PH_c_0000001
  4. 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 Intensity Ladder
 
Probe ID Numbers: PH_c_0000010 to PH_c_0000017

Purpose: To assist in adjusting the scanner settings.

Cy3 Intensity Ladder probes are Cy3-labeled 60-mer oligonucleotide probes designed from an alien sequence that do not cross-hybridize with mouse targets. Eight dilutions, and three to six replicates of each dilution can be excited by a 532 nm(Cy3) laser without hybridization.

Analysis Instructions:
  1. Convert scanned data into Excel format
  2. Sort data by column name 'ID'
  3. Copy ~90 rows of data with IDs above.
  4. 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 probe 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 Probe Concentration Unit
PH_c_0000010 0.39
PH_c_0000011 0.78
PH_c_0000012 1.56
PH_c_0000013 3.125
PH_c_0000014 6.25
PH_c_0000015 12.50
PH_c_0000016 25.00
PH_c_0000017 50.00

Table 1: Mouse OneArray Cy3 Ladder Probe information
ID F532 Median F532 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 Cy3 Ladder Control





Probe ID F532 Median F532 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 Cy3 Ladder Control Output







Figure 3: Sample Cy3 Ladder Control Output


Positive Controls
Intrinsic Hybridization Controls (IHCs)
 
Probe ID Number: PH_c_0000100

Purpose: To monitor the overall quality of sample processing and hybridization efficiency.

IHCs contain a mixture of probes from 11 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_0000101 to PH_c_0000116

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
  1. Convert scanned data into Excel format.
  2. Sort data by column name 'ID'.
  3. Copy rows of data with IDs above.
  4. 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_mc_0000001 to PH_mc_0000014

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 from the 3' end of each transcript.

Analysis Instructions:

  1. Convert scanned data into Excel format.
  2. Sort data by column name ‘ID’.
  3. Copy rows of data with IDs PH_mc_0000001 to PH_mc_0000014.
  4. 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
ITQC_1 TATA box binding protein (Tbp)
ITQC_2 transferrin receptor (Tfrc)
ITQC_4 ribophorin II (Rpn2)
ITQC_6 cathepsin D (Ctsd)
ITQC_7 vimentin (Vim)

Table 4: ITQC Genes


ITQC Gene No. 300-600 nt 900-1200 nt
ITQC_1 PH_mC_0000001 PPH_mC_0000002
ITQC_2 PH_mC_0000003 PH_mC_0000004
ITQC_4 PH_mC_0000007 PH_mC_0000008
ITQC_6 PH_mC_0000011 PH_mC_0000012
ITQC_7 PPH_mC_0000013 PH_mC_0000014
Table 5: ITQC Transcript Locations
Probe ID Sample B
PH_mC_0000001 437.3
PH_mC_0000002 336.5
PH_mC_0000003 2011.7
PH_mC_0000004 665.5
PH_mC_0000007 3253.8
PH_mC_0000008 2516.3
PH_mC_0000011 5186.0
PH_mC_0000012 5556.2
PH_mC_0000013 4395.5
PH_mC_0000014 3362.6

Table 6: Averaged ITQC Intensities

Figure 5: ITQC


Negative Controls
Ambion ArrayControlT 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 ArrayControlT RNA SPIKE #1-8 from Ambion, Inc., has been licensed for use with Mouse 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 mouse genomic probes on the Mouse OneArray DNA microarray.

Analysis Instructions:

  1. Convert scanned data into Excel format
  2. Sort data by column name 'ID'
  3. Copy rows of data with IDs AM_c_0000015 to AM_c_0000022
  4. 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 ArrayControlT 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 ArrayControlT 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 mouse genomic probes on Mouse 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:
  1. Convert scanned data into Excel format
  2. Sort data by column name 'ID'
  3. Copy rows of data with IDs PH_c_0000066 to PH_c_0000071
  4. 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 ArrayControlT 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
Sample A
Sample B
PH_c_0000066 36 37
PH_c_0000067 38 60
PH_c_0000068 40 52
PH_c_0000069 39 40
PH_c_0000070 41 41
PH_c_0000071 40 41

Table 10: ETQC Sample Data
Figure 7: ETQC Sample Output