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Development of a microarray chip for gene expression in rabbit ocular research.

Popp MP, Liu L, Timmers A, Esson DW, Shiroma L, Meyers C, Berceli S, Tao M, Wistow G, Schultz GS, Sherwood MB - Mol. Vis. (2007)

Bottom Line: The expression of 315 genes was significantly altered by glaucoma filtration surgery.Genes whose expression was altered included proteins associated with inflammatory response, defense response, and proteins involved in synthesis of the extracellular matrix.The results of this rabbit microarray study are consistent with those from other wound healing studies, indicating that this array can provide valid information on broad patterns of gene expression.

View Article: PubMed Central - PubMed

Affiliation: University of Florida's Interdisciplinary Center for Biotechnology Research, University of Florida, Gainsville, FL 32610-0294, USA. mpopp@obgyn.ufl.edu <mpopp@obgyn.ufl.edu>

ABSTRACT

Purpose: To develop a microarray for the rabbit that can be used for ocular gene expression research.

Methods: Messenger RNA was isolated from anterior segment tissues (cornea, conjunctiva, and iris) and posterior segment tissues (lens, retina, and sclera) of rabbit eyes and used to create two independent cDNA libraries through the NEIBank project. Clones from each of these libraries were sequenced from both the 5' and 3' ends. These sequences and those from the National Center for Biotechnology Information (NCBI) taxonomy database for rabbit were combined and electronically assembled into a set of unique nonoverlapping continuous sequences (contigs). For each contig, a homology search was performed using BLASTX and BLASTN against both the NCBI NR and NT databases to provide gene annotation. Unique contigs were sent to Agilent Technologies, where 60 base oligonucleotide probes were designed and synthesized, in situ, on two different arrays in an 8 array x 1900 element format. Glaucoma filtration surgery was performed on one eye of six rabbits. After 14 days, tissue was harvested from the conjunctiva and Tenon's capsule of both the surgically treated and untreated control eyes. Total RNA from each sample was labeled with cyanine dyes and hybridized to our custom microarrays.

Results: Of the 3,154 total probes present on the two arrays, 2,522 had a signal value above the background. The expression of 315 genes was significantly altered by glaucoma filtration surgery. Genes whose expression was altered included proteins associated with inflammatory response, defense response, and proteins involved in synthesis of the extracellular matrix.

Conclusions: The results of this rabbit microarray study are consistent with those from other wound healing studies, indicating that this array can provide valid information on broad patterns of gene expression. This is the first microarray available for rabbit studies and is a valuable tool that can be used to study molecular events in the eye.

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Related in: MedlinePlus

Examples of images generated from scanned microarrays. Example of an image generated from a scanned eight-pack microarray (A) and a single 1,900 element (B) microarray. Each circle represents a unique spotted probe. Red probes indicate that gene expression in the surgically treated eye is higher than in the control, while green marks higher gene expression in the control. Yellow indicates no difference in gene expression between the surgically treated and control eyes. Black denotes absence of detectable signal.
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f1: Examples of images generated from scanned microarrays. Example of an image generated from a scanned eight-pack microarray (A) and a single 1,900 element (B) microarray. Each circle represents a unique spotted probe. Red probes indicate that gene expression in the surgically treated eye is higher than in the control, while green marks higher gene expression in the control. Yellow indicates no difference in gene expression between the surgically treated and control eyes. Black denotes absence of detectable signal.

Mentions: An example of a scanned slide is shown in Figure 1. Of the 3,154 total probes present on the two arrays, 2,522 had a signal value above the background, in either the red or green channel, on at least two experimental arrays. Genes represented by these probes were considered present. The remainder were considered absent (no expression under any experimental condition). However, some of the absent calls may be attributed to "bad" probes which have suboptimal hybridization characteristics and may not work under any experimental conditions. Only future hybridizations under different experimental conditions will allow us to determine which probes need to be redesigned.


Development of a microarray chip for gene expression in rabbit ocular research.

Popp MP, Liu L, Timmers A, Esson DW, Shiroma L, Meyers C, Berceli S, Tao M, Wistow G, Schultz GS, Sherwood MB - Mol. Vis. (2007)

Examples of images generated from scanned microarrays. Example of an image generated from a scanned eight-pack microarray (A) and a single 1,900 element (B) microarray. Each circle represents a unique spotted probe. Red probes indicate that gene expression in the surgically treated eye is higher than in the control, while green marks higher gene expression in the control. Yellow indicates no difference in gene expression between the surgically treated and control eyes. Black denotes absence of detectable signal.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2536532&req=5

f1: Examples of images generated from scanned microarrays. Example of an image generated from a scanned eight-pack microarray (A) and a single 1,900 element (B) microarray. Each circle represents a unique spotted probe. Red probes indicate that gene expression in the surgically treated eye is higher than in the control, while green marks higher gene expression in the control. Yellow indicates no difference in gene expression between the surgically treated and control eyes. Black denotes absence of detectable signal.
Mentions: An example of a scanned slide is shown in Figure 1. Of the 3,154 total probes present on the two arrays, 2,522 had a signal value above the background, in either the red or green channel, on at least two experimental arrays. Genes represented by these probes were considered present. The remainder were considered absent (no expression under any experimental condition). However, some of the absent calls may be attributed to "bad" probes which have suboptimal hybridization characteristics and may not work under any experimental conditions. Only future hybridizations under different experimental conditions will allow us to determine which probes need to be redesigned.

Bottom Line: The expression of 315 genes was significantly altered by glaucoma filtration surgery.Genes whose expression was altered included proteins associated with inflammatory response, defense response, and proteins involved in synthesis of the extracellular matrix.The results of this rabbit microarray study are consistent with those from other wound healing studies, indicating that this array can provide valid information on broad patterns of gene expression.

View Article: PubMed Central - PubMed

Affiliation: University of Florida's Interdisciplinary Center for Biotechnology Research, University of Florida, Gainsville, FL 32610-0294, USA. mpopp@obgyn.ufl.edu <mpopp@obgyn.ufl.edu>

ABSTRACT

Purpose: To develop a microarray for the rabbit that can be used for ocular gene expression research.

Methods: Messenger RNA was isolated from anterior segment tissues (cornea, conjunctiva, and iris) and posterior segment tissues (lens, retina, and sclera) of rabbit eyes and used to create two independent cDNA libraries through the NEIBank project. Clones from each of these libraries were sequenced from both the 5' and 3' ends. These sequences and those from the National Center for Biotechnology Information (NCBI) taxonomy database for rabbit were combined and electronically assembled into a set of unique nonoverlapping continuous sequences (contigs). For each contig, a homology search was performed using BLASTX and BLASTN against both the NCBI NR and NT databases to provide gene annotation. Unique contigs were sent to Agilent Technologies, where 60 base oligonucleotide probes were designed and synthesized, in situ, on two different arrays in an 8 array x 1900 element format. Glaucoma filtration surgery was performed on one eye of six rabbits. After 14 days, tissue was harvested from the conjunctiva and Tenon's capsule of both the surgically treated and untreated control eyes. Total RNA from each sample was labeled with cyanine dyes and hybridized to our custom microarrays.

Results: Of the 3,154 total probes present on the two arrays, 2,522 had a signal value above the background. The expression of 315 genes was significantly altered by glaucoma filtration surgery. Genes whose expression was altered included proteins associated with inflammatory response, defense response, and proteins involved in synthesis of the extracellular matrix.

Conclusions: The results of this rabbit microarray study are consistent with those from other wound healing studies, indicating that this array can provide valid information on broad patterns of gene expression. This is the first microarray available for rabbit studies and is a valuable tool that can be used to study molecular events in the eye.

Show MeSH
Related in: MedlinePlus