Yeast OneArray® Controls
Orientation Settings and Controls

Dye Intensity Controls

Positive Controls

Negative Controls

Extrinsic Target Quality Control (ETQC)
Below is a brief description of the Yeast 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 Yeast 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 Yeast 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: Yeast OneArray OGAM and CGAM Positions


Figure 2: Yeast 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 Yeast 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 Yeast 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: Yeast 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

Phalanx Probe Index

Gene Name

Description

YLL039C_01

Operon positive control_UBI4

Ubiquitin, becomes conjugated to proteins, marking them for selective degradation via the ubiquitin-26S proteasome system; essential for the cellular stress response.

YBL092W_01

Operon positive control_RPL32

Protein component of the large (60S) ribosomal subunit, has similarity to rat L32 ribosomal protein; overexpression disrupts telomeric silencing.

YBR181C_01

Operon positive control_RPS6A

Protein component of the small (40S) ribosomal subunit; identical to Rps6Ap and has similarity to rat S6 ribosomal protein.

YER103W_01

Operon positive control_SSA4

Heat shock protein that is highly induced upon stress; plays a role in SRP-dependent cotranslational protein-membrane targeting and translocation; member of the HSP70 family; cytoplasmic protein that concentrates in nuclei upon starvation.

YFL039C_01

Operon positive control_ACT1

Actin, structural protein involved in cell polarization, endocytosis, and other cytoskeletal functions.

YIL115C_01

Operon positive control_NUP159

Subunit of the nuclear pore complex that is found exclusively on the cytoplasmic side, forms a subcomplex with Nup82p and Nsp1p, required for mRNA export.

YJL052W_01

Operon positive control_TDH1

Glyceraldehyde-3-phosphate dehydrogenase, isozyme 1, involved in glycolysis and gluconeogenesis; tetramer that catalyzes the reaction of glyceraldehyde-3-phosphate to 1,3 bis-phosphoglycerate; detected in the cytoplasm and cell-wall.

YJL061W_01

Operon positive control_NUP82

Subunit of the nuclear pore complex (NPC), forms a subcomplex with Nup159p and Nsp1p, interacts with Nup116p and is required for proper localization of Nup116p in the NPC.

YJR009C_01

Operon positive control_TDH2

Glyceraldehyde-3-phosphate dehydrogenase, isozyme 2, involved in glycolysis and gluconeogenesis; tetramer that catalyzes the reaction of glyceraldehyde-3-phosphate to 1,3 bis-phosphoglycerate; detected in the cytoplasm and cell-wall.

YLL026W_01

Operon positive control_HSP104

Heat shock protein that cooperates with Ydj1p (Hsp40) and Ssa1p (Hsp70) to refold and reactivate previously denatured, aggregated proteins; responsive to stresses including: heat, ethanol, and sodium arsenite; involved in [PSI+] propagation.

YLR212C_01

Operon positive control_TUB4

Gamma-tubulin, involved in nucleating microtubules from both the cytoplasmic and nuclear faces of the spindle pole body.

YPL016W_01

Operon positive control_SWI1

Subunit of the SWI/SNF chromatin remodeling complex, which regulates transcription by remodeling chromosomes; required for transcription of many genes, including ADH1, ADH2, GAL1, HO, INO1 and SUC2.


Negative Controls

Phalanx Probe Index

Gene Name

Description

YCONTROL02

Operon Negative control_1

Randomly_generated_negative_control

YCONTROL04

Operon Negative control_2

Randomly_generated_negative_control

YCONTROL19

Operon Negative control_3

Randomly_generated_negative_control

YCONTROL57

Operon Negative control_4

Randomly_generated_negative_control

YCONTROL60

Operon Negative control_5

Randomly_generated_negative_control

YCONTROL63

Operon Negative control_6

Randomly_generated_negative_control

YCONTROL77

Operon Negative control_7

Randomly_generated_negative_control

YCONTROL91

Operon Negative control_8

Randomly_generated_negative_control

YCONTROL97

Operon Negative control_9

Randomly_generated_negative_control

YCONTROL98

Operon Negative control_10

Randomly_generated_negative_control

YCONTROL99

Operon Negative control_11

Randomly_generated_negative_control

YCONTROLAF

Operon Negative control_12

Randomly_generated_negative_control


Extrinsic Target Quality Control (ETQC)
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 Yeast 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 Yeast genomic probes on the Yeast 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 # Database Reference
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 4: 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 5: Ambion ArrayControl® RNA SPIKES Sample Averaged Intensities


Figure 4: Ambion ArrayControl™ RNA SPIKES Sample Output
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 Yeast genomic probes on Yeast 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 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 6: 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 7: ETQC Sample Data

Figure 5: ETQC Sample Output