Microarray Project:: Reproducibility Data

Table of Contents:

The 605 Endosperm microarrays comprised 8064 ESTs spotted as adjacent duplicate elements. In addition, a panel of 93 control elements (see the control table) was spotted as duplicate elements at two locations (i.e. four times), the two duplicate positions being spaced by 9 mm. The 605 Library (plus controls) was printed in a single array with two replicate spots of each element--replicate spots were printed side-by-side. The 606 Endosperm microarrays comprised 4980 ESTs spotted as adjacent triplicate elements. The same panel of controls (plus several additional controls) was spotted as triplicate elements at two locations (i.e. six times), the two duplicate positions again being spaced by 9 mm.

In order to evaluate the reproducibility of hybridization signals between the replicate elements on the same microarray a 605 Endosperm and a 606 Ear Tisuue microarray were hybridized separately with fluorescent targets derived from endosperm and immature ear, respectively. The 605 Endosperm Microarray was hybridized with labeled polyA+ mRNA derived from OH43 endosperm (14 DAP) —identical in genotype and similar in developmental stage to the polyA+ mRNA used to make the 605 EST library. The 606 Immature Ear Microarray was hybridized with labeled polyA+ mRNA derived from OH43 immature ear (6 cm)—identical in genotype, but slightly older in developmental stage than the polyA+ mRNA used to make the 606 EST library. Simple linear correlation analysis was used to compare the signal intensities of the two replicate spots on the same slide. For the 606 Ear Tissue Microarray (with 3 replicate spots) the signal intensities for the first and third spot were compared. Pearson correlation coefficients ranging from 0.96-0.98 indicate a high degree of reproducibility in hybridization among replicate spots on the same slide for both the 605 and 606 microarrays.

In order to evaluate the reproducibility of hybridization signals between the same elements on replicate microarrays, two 605 Endosperm and two 606 Ear Tissue microarrays were hybridized using fluorescent targets derived from both endosperm and immature ear (OH43 endosperm (14 DAP) and OH43 immature ear (2 cm)). Each microarray was hybridized with both targets labeled separately with either Cy3 or Cy5 dyes; dyes were reversed for the two microarrays from each EST library. Linear correlation analysis was used to compare signal intensities of hybridization to each element by the same target on two different slides. Log10 transformed ratios of hybridization signals for both targets on two different slides were also compared. Pearson correlation coefficients ranging from 0.77 to 0.87 indicate the reproducibility in hybridization between replicate Microarrays. As expected, correlation coefficients demonstrated that hybridization was more variable between slides than between replicates.

Microarray Format Details

The following documents describe the various microarray formats used to produce the data below.

486 Microarray
605-3 and 605-4-1,2 Microarray
606 Microarray
614 Microarray
Unigene 1 Microarray
Master Unigene 1

(Other formats: Bitmap. Warning: This file is very big.)

605 Endosperm Microarray hybridized with labeled polyA+ mRNA derived from OH43 endosperm (14 DAP). Signal intensity values ranging from low to high are represented by color changes from blue to green, yellow, red and white, respectively. Black areas have no detectable hybridization signal. Signal intensity data for this image can be downloaded in text format.

605 Microarray Image 1 Table Legend:

The 605 Endosperm Microarrays are composed of eight subarrays arranged in 4 rows and 2 columns. Each of the 8 subarrays is composed of a 46 x 46 matrix of spots printed at a spacing of 190 um with a 380 um gap between subarrays. Each DNA element on the slide can be located by knowing the Subarray Row and Subarray Column and the Row and Column within the subarray where the element is printed. ESTs can be identified by Stanford Id (number based on the EST project/plate/and platewell where EST is located) and by GenBank Accession Number. Control sequences have sequence names and descriptors in place of Stanford ID and GenBank Accession Number (for more information on controls—see the Control Table page.) For each element on the Microarray, the median signal, the median value of the local background, and the net signal intensity are given (signal-background). For more information about the format of the 605 Microarrays visit the information button on the Microarray Elements Search page.

605 Endosperm Microarray Graph 1: Reproducibility of hybridization signal between side-by-side replicate spots on the same slide. Graph compares hybridization intensity values of replicate 1 versus replicate 2. Low signal intensity values were corrected by setting them equal to two times the mean signal background (local background for all elements). Saturated signal intensity values are not shown.

(Other formats: Tiff. Warning: This file is huge.)

605 Endosperm Microarray Image 2:

605 Endosperm Microarray hybridized with labeled targets derived from OH43 endosperm (14 DAP) and OH43 immature ear (2 cm). Image is a false color overlay: red, green, and yellow designate elements with lesser, greater or equal hybridization by ear derived targets relative to endosperm derived targets, respectively. Signal intensity data for Image 2 can be downloaded in text format.

605 Microarray Image 2 Table Legend:

The 605 Endosperm Microarrays are composed of eight subarrays arranged in 4 rows and 2 columns. Each of the 8 subarrays is composed of a 46 x 46 matrix of spots printed at a spacing of 190 um with a 380 um gap between subarrays. Each DNA element on the slide can be located by knowing the Subarray Row and Subarray Column and the Row and Column within the subarray where the element is printed. ESTs can be identified by Stanford Id (number based on the EST project/plate/and platewell where EST is located) and by GenBank Accession Number. Control sequences have sequence names and descriptors in place of Stanford ID and GenBank Accession Number (for more information on controls—see the Control Table page.) Hybridization signal intensity data are given for both endosperm and ear tissue derived targets on two different microarrays. For each element on the Microarray, the median signal, the median of the local background, and the net signal intensity are given (signal-background). For more information about the format of the 605 Microarrays visit the information button on the Microarray Elements Search page.

 

605 Microarray Graph 2: Reproducibility hybridization signal intensity between replicate microarrays. Mean signal intensity was calculated for replicate spots on each of two replicate microarrays. Graph compares the mean signal intensity values on microarray 1 versus microarray 2. Low signal intensity values were corrected by setting them equal to two times the mean signal background (local background for all elements). Saturated signal intensity values are not shown.

605 Microarray Graph 3: Reproducibility of immature ear:endosperm signal intensity ratios between microarrays. Ratios of mean signal intensities (mean of two replicate spots) on each of two replicate microarrays were calculated and transformed (log10 transformation). Graph compares the log10 transformed values of mean ratio on microarray 1 versus microarray 2.

(Other formats: Bitmap | Tiff. Warning: These files are huge.)

606 Immature Ear Microarray hybridized with labeled polyA+ mRNA derived from OH43 immature ear (6 cm). Signal intensity ranges from low to high are represented by color changes from blue to green, yellow, red and white, respectively. Black areas have not detectable hybridization. Signal intensity data for this image can be downloaded in text format.

606 Immature Ear Microarray Image 1 Table Legend: The 606 Ear Tissue Microarrays are composed of eight subarrays arranged in 4 rows and 2 columns. Each of the 8 subarrays is composed of a 45 x 45 matrix of spots printed at a spacing of 195 um with a 250 um gap between subarrays. Each DNA element on the slide can be located by knowing the Subarray Row and Subarray Column and the Row and Column within the subarray where the element is printed. ESTs can be identified by Stanford Id (number based on the EST project/plate/and platewell where EST is located) and by GenBank Accession Number. Control sequences have sequence names and descriptors in place of Stanford ID and GenBank Accession Number (for more information on controls—see the Control Table page.) For each spot on the Microarray, the median signal, the median of the local background, and the net signal intensity are given (signal-background). For more information about the format of the 606 Microarrays visit the information button on the Microarray Elements Search page.

606 Ear Tissue Microarray Graph 1: Reproducibility of hybridization signal between side-by-side replicate spots on the same slide. Graph compares hybridization intensity values of replicate 1 versus replicate 3. Low signal intensity values were corrected by setting them equal to two times the mean signal background (local background for all elements). Saturated signal intensity values are not shown.

(Other formats: Tiff. Warning: This file is huge.)

606 Immature Ear Microarray Image 2:

606 Immature Ear Microarray hybridized with labeled polyA+ mRNA derived from OH43 endosperm (14 DAP) and OH43 immature ear (2 cm). Image is a false color overlay: red, green, and yellow designate elements with lesser, greater, or equal hybridization by endosperm derived targets relative to ear derived targets, respectively. Signal intensity data for Image 2 can be downloaded in text format.

606 Microarray Image 2 Table Legend:

The 606 Ear Tissue Microarrays are composed of eight subarrays arranged in 4 rows and 2 columns. Each of the 8 subarrays is composed of a 45 x 45 matrix of spots printed at a spacing of 195 um with a 250 um gap between subarrays. Each DNA element on the slide can be located by knowing the Subarray Row and Subarray Column and the Row and Column within the subarray where the element is printed. ESTs can be identified by Stanford Id (number based on the EST project/plate/and platewell where EST is located) and by GenBank Accession Number. Control sequences have sequence names and descriptors in place of Stanford ID and GenBank Accession Number (for more information on controls—see the Control Table page.) Hybridization signal intensity data are given for both ear tissue and endosperm derived targets on two different microarrays. For each element on the Microarray, the median signal, the median of the local background, and the net signal intensity are given (signal-background). For more information about the format of the 606 Microarrays visit the information button on the Microarray Elements Search page.

606 Microarray Graph 2: Reproducibility hybridization signal intensity between replicate microarrays. Mean signal intensity was calculated for replicate spots on each of two replicate microarrays. Graph compares the mean signal intensity values on microarray 1 versus microarray 2. Low signal intensity values were corrected by setting them equal to two times the mean signal background (local background for all elements). Saturated signal intensity values are not shown.

606 Microarray Graph 3: Reproducibility of endosperm:ear signal intensity ratios between microarrays. Ratios of mean signal intensities (mean of two replicate spots) on each of two replicate microarrays were calculated and transformed (log10 transformation). Graph compares the log10 transformed values of mean ratio on microarray 1 versus microarray 2..

 

(Other formats: Bitmap | Tiff. Warning: These files are huge.)

Figure Legend: 614 Root Microarray hybridized with labeled polyA+ mRNA derived from B73 root (7 day). Signal intensity values ranging from low to high are represented by color changes from blue to green, yellow, red and white, respectively. Black areas have no detectable hybridization signal. Signal intensity data for this image will soon be available text format.

614 Root Microarray Graph 1

Excel format

Reproducibility of hybridization signal between side-by-side triplicate spots on the same slide. Graph compares hybridization intensity values of spot 1 versus spot 3.

486 Immature Leaf Microarray

486 Immature Leaf Microarray Image 1

TIF (this is a big file)

Microarray hybridized with labeled polyA+ mRNA derived from B73 immature leaves (8 leaf stage). Signal intensity values ranging from low to high are represented by color changes from blue to green, yellow, red and white, respectively. Black areas have no detectable hybridization signal.

486 Immature Leaf Microarray Graph 1

Excel format

Reproducibility of hybridization signal between side-by-side triplicate spots on the same slide. Graph compares hybridization intensity values of spot 1 versus spot 3.

486 Immature Leaf Microarray Additional Data

This Excel spreadsheet contains additional data on the 486 immature leaf microarray.

Unigene-1 Microarray

Unigene-1 Microarray Image 1

TIF (this is a big file)

Unigene 1-1-01 hybridized with labeled polyA+ mRNA derived from OH43 Husk tissue.

Data in Excel format (.xls)

Reproducibility of hybridization signal intensity between replicate spots on the same slide. Signal intensity data for Graph 1 and its data can be downloaded in Excel format (you need Microsoft Excel to open it).

This Excel spreadsheet contains additional data on the 486 immature leaf microarray.

Unigene 1-2 Microarray

Unigene 1-2 Microarray Image 1

TIF (this is a big file)

Unigene 1-2-31 hybridized with labeled polyA+ mRNA derived from the endosperm.

Data in Excel format (.xls)

Reproducibility of hybridization signal intensity between replicate spots on the same slide. Signal intensity data for Graph 1 and its data can be downloaded in Excel format (you need Microsoft Excel to open it).

Master Unigene 1 Microarray

Master Unigene 1 Microarray Image 1

BMP (this is a big file)

MGD-MUG-01-066 Master unigene array hybridized with labeled polyA+ mRNA derived from 5 day old B73 seedlings. Signal intensity ranges from low to high are represented by color changes from blue to green, yellow, red and white, respectively. Black areas have not detectable hybridization. Signal intensity data for this image can be downloaded in text format.