OneArray® Rice Microarray Controls

Orientation Settings and Controls

Dye Intensity Controls

Positive Controls

Negative Controls 		Below is a brief description of the Rice 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 rice 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 rice 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: Rice OneArray® OGAM and CGAM Positions


Figure 2: Rice 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 rice 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 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 rice 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:
  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 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: Rice OneArray® Cy3 and Cy5 Ladder Probe information
Probe ID F635
Median 		F635
Mean
PH_c_0000010 520 526
PH_c_0000011 2424 2504
PH_c_0000012 8366 8677
PH_c_0000013 21476 23020
PH_c_0000014 43794 42831
PH_c_0000015 59997 62890
PH_c_0000016 65535 65535
PH_c_0000017 65535 65535

Table 2: Sample Cy5 Ladder Control 		RiOA_ladder

Figure 3: Sample Cy5 Ladder Control Output


Positive Controls
Intrinsic Hybridization Controls (IHCs)
 
Probe ID Number: PH_Cos_0000013

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

IHCs contain a mixture of probes from 33 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_Cos_0000014 to PH_Cos_0000021

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_Cos_0000001 to PH_Cos_0000006

Purpose: To monitor the length of the targets for assessment of the quality of sample integrity and processing.

Two probes per gene were selected from three robustly expressed housekeeping genes. For each gene, one probe is designed from regions of 300-600 bp, and 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_Cos_0000001 to PH_Cos_0000006.
  4. Obtain the mean of the 3-6 replicates signals in the '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 LOC_Os02g46510.1 13102.m05223 cDNA adaptor complexes medium subunit family domain containing protein
ITQC_2 LOC_Os02g46510.1 13102.m05223 cDNA adaptor complexes medium subunit family domain containing protein
ITQC_3 LOC_Os03g21210.1 13103.m02541 cDNA endoglucanase

Table 4: ITQC Genes


ITQC Gene No. 300-600 nt(S1) 900-1200 nt(S2)
ITQC_1 PH_Cos_0000001 PH_Cos_0000002
ITQC_2 PH_Cos_0000003 PH_Cos_0000004
ITQC_3 PH_Cos_0000005 PH_Cos_0000006
Table 5: ITQC Transcript Locations


Figure 5: ITQC Ratio Output


Negative Controls
Ambion ArrayControlT
RNA SPIKE #3-7
 
Probe ID Numbers: AM_c_0000017 to AM_c_0000021

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 #3-7 from Ambion, Inc., has been licensed for use with Rice 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 rice genomic probes on the Rice 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_0000017 to AM_c_0000021
  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 ArrayControlT RNA SPIKE ID #
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

Table 7: Ambion ArrayControlT RNA SPIKES


Probe ID # Shoot Root
AM_c_0000017 105.8 99.2
AM_c_0000018 55.2 94.3
AM_c_0000019 226.3 298.0
AM_c_0000020 91.5 83.7
AM_c_0000021 71.7 60.7

Table 8: Ambion ArrayControlT RNA SPIKES
Sample Averaged Intensities


Figure 6: Ambion ArrayControlT RNA SPIKES Sample Output
Extrinsic Target Quality Control (ETQC)
 
Probe ID Numbers: mCN** and PH_cc_**

Purpose: To monitor the specificity of hybridization.

ETQC probes are alien probe sequences that do not cross-hybridize with rice genomic probes on Rice 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 mCN** and PH_cc_**
  4. Obtain the mean of the 3-6 replicates signals in the '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.


Figure 7: ETQC Sample Output