Microarray Analysis of Gene Expression in Human Donor Sclera
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Molecular Vision 2004; 10:163-76 <http://www.molvis.org/molvis/v10/a22> ©2004 Molecular Vision Received 3 October 2003 | Accepted 16 March 2004 | Published 22 March 2004 Microarray analysis of gene expression in human donor sclera Terri L. Young,1,2 Genaro S. Scavello,1,2 Prasuna C. Paluru,1,2 Jonathan D. Choi,2,3 Eric F. Rappaport,2 Jody A. Rada4 Divisions of 1Ophthalmology and 2Genetics, Children’s Hospital of Philadelphia and the University of Pennsylvania, Philadelphia, PA; 3Department of Medical and Molecular Genetics, GKT School of Medicine, London; 4Department of Cell Biology, University of Oklahoma Health Science Center, Oklahoma City, OK Purpose: To develop gene expression profiles of human sclera to allow for the identification of novel, uncharacterized genes in this tissue-type, and to identify candidate genes for scleral disorders. Methods: Total RNA was isolated from 6 donor sources of human sclerae, and reverse transcribed into cDNA using a T7- (dT) 24 primer. The resulting cDNA was in vitro transcribed to produce biotin-labeled cRNA, fragmented, and mixed with hybridization controls before a 16 h hybridization step with oligonucleotide probes on 6 Affymetrix U95A chips. The chips were scanned twice at 570 nM and the data collected using GeneChip software. Array analyses were carried out with Microarray Suite, version 5.0 (Affymetrix), using the expression analysis algorithm to run an absolute analysis after cell intensities were computed. All arrays were scaled to the same target intensity using all probe sets. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to validate the microarray results. Results: There were 3,751 genes with “present” calls assigned independently to all six human scleral samples. These genes could be clustered into 4 major categories; transcription (10%), metabolism (8.8%), cell growth and proliferation (5.4%), and extracellular matrix (2%). Many extracellular matrix proteins, such as collagens 6A3 and 10A1, thrombospondins 2 and 4, and dystroglycan have not previously been shown to be expressed in sclera. RT-PCR results confirmed scleral expression in 7 extracellular matrix genes examined. Conclusions: This study demonstrated the utility of gene microarray technology in identifying global patterns of scleral gene expression, and provides an extended list of genes expressed in human sclera. Identification of genes expressed in sclera contributes to our understanding of scleral biology, and potentially provides positional candidate genes for scleral disorders such as high myopia. The sclera, the tough outer wall of the eye, is a special- expression for hundreds or thousands of genes in a single ex- ized connective tissue that provides the structural framework periment. that defines the shape (such as the axial length) of the eye. It Microarray expression analysis has features that make it consists largely of collagenous lamellae in close association the most widely used method for profiling mRNA expression. with proteoglycans and glycoproteins [1-5]. Changes in the DNA segments representing the collection of genes to be as- extracellular matrix components of the sclera or in molecules sayed are amplified by polymerase chain reaction (PCR) and required for the synthesis and degradation of the scleral ma- mechanically spotted at high density on glass microscope slides trix may lead to significant changes in scleral biomechanical or nylon membranes using robotic systems. Experimental properties, leading to changes in scleral shape, ocular size and mRNA is labeled as a complex mixture and exposed to the therefore the refractive state of the eye [6-10]. microarray. Labeled mRNA will bind to complementary se- The profiling of gene expression in specific tissues pro- quences on the microarray and can be detected in a semi-quan- vides useful information to characterize gene function and tis- titative manner using automated techniques. The microrarrays sue physiology [11-13]. This baseline knowledge should fa- are queried in a co-hybridization assay using fluorescently cilitate the identification of alterations from normal gene ex- labeled probes prepared from mRNA from the cellular pheno- pression that play important roles in disease pathogenesis. Prior types of interest. The kinetics of hybridization allows relative research on the human sclera has focused on the detection of expression levels to be determined based on the ratio with individual proteins of interest using techniques such as im- which each probe hybridizes to an individual array element. munohistochemistry and in situ hybridization. Due to the rapid Hybridization is assayed using a confocal laser scanner to progress of the Human Genome Project and the development measure fluorescence intensities, which allows the simulta- of high-throughput techniques such as cDNA microarray analy- neous determination of the relative expression levels of all the sis [14-16], messenger RNA (mRNA) expression can be de- genes represented in the array [16-19]. termined on a global scale with parallel assessment of gene Because of the success of microarray analysis use in other biological systems and other eye tissue types [16-23], we Correspondence to: Terri L. Young, M.D., Division of Ophthalmol- sought to apply this technology to study gene expression in th ogy, Children’s Hospital of Philadelphia, 34 and Civic Center Bou- human donor scleral tissue. This analysis provides baseline levard, Philadelphia, PA, 19104; Phone: (215) 590-9950; FAX: (215) expression information regarding the genetic basis of normal 590-3850; email: [email protected] 163 Molecular Vision 2004; 10:163-76 <http://www.molvis.org/molvis/v10/a22> ©2004 Molecular Vision scleral function, as well as for scleral disease processes such Target was prepared using 5-20 µg of total RNA for each as pathologic myopia with excess axial elongation, mi- donor sclera. First strand cDNA was synthesized using Super- crophthalmia, scleral ectasia, focal staphylomatous formation, script II Reverse Transcriptase (Invitrogen Corporation, and inflammation. Knowledge of genes expressed or not ex- Carlsbad, CA) and a T7-(dT)24 primer to incorporate the T7 pressed in a particular scleral disorder could lead to novel and priming site into the cDNA. Following RNA degradation with definitive treatment strategies, such as interventional or gene RNase H and second strand cDNA synthesis with DNA poly- therapies. These strategies may be particularly relevant for the merase I, the double-stranded cDNA was extracted with sclera because the tissue is relatively less complex, can be phenol:chlorform:isoamyl alcohol (25:24:1). Approximately manipulated ex vivo, and can be readily assessed visually. 1 µg of cDNA was used as template in an in vitro transcrip- tion assay reaction (Enzo Life Sciences, Inc., Farmingdale, METHODS NY) that incorporates biotin into the resulting cRNA. The Affymetrix prefabricated chips: Each probe set on an cRNA was fragmented to a size range of 35-200 bases prior to Affymetrix chip is represented by multiple features or probes use in hybridization by incubation at 94 °C for 35 min in frag- consisting of synthesized oligonucleotides. Half of the fea- mentation buffer (40 mM Tris acetate, pH 8.1, 125 mM KOAc, tures are exact match representations at different positions 30 mM MgOAc). Fifteen µg of fragmented probe was mixed along the length of the expressed regions of the gene. The with hybridization controls, herring sperm DNA (final con- other half contains the same oligonucleotide probes, but with centration 100 µg/ml), and acetylated BSA (final concentra- a single mismatch in the middle. The range of 22-40 cells on tion 100 µg/ml) in hybridization buffer (100 mM MES, 1 M the HG-U95A chip includes some controls for which there is [Na+], 20 mM EDTA, 0.01% Tween 20). The hybridization a smaller or larger number than the 32 used as the probe sets mixture was heated at 99 °C for five min, incubated at 45 °C for experimental genes. On the Affymetrix HG-U95A chip, for five min, and centrifuged at 13,000x g for 5 min. Test 3 for any given experimental gene there are 16 positions at which chips were pre-hybridized with 80 µl of 1X hybridization buffer hybridization can be assessed, and the comparison between for 10 min at 45 °C and 60 RPM in the hybridization oven. the perfect match and mismatch probes provides a control for Following removal of the pre-hybridization buffer, the 6 non-specific hybridization. The Affymetrix HG-U95A chip Affymetrix chips were filled with 200 µl of the hybridization contains 12,626 probe sets with some redundancy for certain mixture and incubated at 45 °C and 60 RPM for 16 h [26,27]. genes or splice variants. Hybridization mixture was removed and saved, and each Target preparation, hybridization, and washing: Total chip was filled with 250 µl of non-stringent wash buffer (6X RNA was isolated from the sclera of 6 human donor eyes. The SSPE, 0.01% Tween 20). Further washing and staining of the eyes were from both male and female donors, ages 35-68 years. chips was conducted on the fluidics station with non-stringent The human eyes were obtained from the Lions Eye Bank of washing buffer, stringent washing buffer (100 mM MES, 0.1 Minnesota under an approved Institutional Review Board pro- M [Na+], 0.01% Tween 20), and stain buffer (100 mM MES, 1 tocol at the University of Minnesota. The procurement and M [Na+], 0.05% Tween 20) containing 10 µg /ml of streptavidin use of human tissues was in compliance with the tenets of the phycoerythrin (SAPE, Molecular Probes, Inc., Eugene, OR). Declaration of Helsinki. The donor eyes were obtained as ei- The signal was amplified by an additional treatment with goat ther whole globes or posterior poles with the cornea removed. IgG (0.1 mg/ml), biotinylated antibody (3 µg/ml) and a sec- The eyes were treated by submersion in RNALater solution ond staining with SAPE.