Abstract
The R28 rat retinal progenitor cell line was developed from postnatal day six rat retina immortalized with the 12S E1A gene of adenovirus. R28 cells have been distributed to over 100 laboratories worldwide, with over 60 publications on topics that include in vitro toxicology, cellular physiology, gene expression analysis, and experimental transplantation. In this paper, we present a microarray dataset of R28 cells that describes the presence or absence of 8799 genes and ESTs that may be relevant to current and future studies of R28 retinal precursor cells.
1. Introduction
R28 cells were developed over 15 years ago as a growth-stimulated, nontumorigenic retinal cell line immortalized by the 12S E1A gene of adenovirus [1]. Over the years, these cells have been utilized for a wide range of studies that include in vitro toxicology [2], neuroprotection [3, 4], light responses [5], diabetic complications [6], and retinal transplantation [7]. An abbreviated list of genes and ESTs from this R28 cell line dataset with particular relevance to neuroscience was described previously [8]. The presence of a subset of genes (e.g., MAP2, calbindin, nestin, and syntaxin) was validated by immunostaining, while functionality was measured by electrophysiological responses to dopamine, serotonin, and muscarinic receptor agonists [8]. Here, we present the entire 8799 gene/EST microarray dataset in open access format for the benefit of neuroscience investigators with an interest in retinal cell biology.
2. Methodology
R28 cells were derived by three rounds of limiting dilution of the parental E1A-NR.3 cell line. Parental E1A-NR.3 cells were originally developed from postnatal day 6 Sprague-Dawley rat retinal tissue immortalized with the 12S E1A gene of adenovirus from a replication-incompetent retroviral vector [1]. Animal sacrifice and tissue harvesting were performed according to institutionally approved animal care and use protocols. R28 cells were grown in Dulbecco’s Modified Eagle’s Medium with 10% calf serum, 0.37% sodium bicarbonate, 0.058% l-glutamine, and 100 μg/mL gentamicin. For this microarray study, R28 cells were trypsinized at passage 48, centrifuged into a pellet, snap-frozen, and stored at −80°C prior to analysis.
Microarray analysis was performed as described previously [8]. Briefly, the RG U34A cDNA microarray (Affymetrix, Inc. Santa Clara, CA, USA), a rat microarray that contains 8799 genes and ESTs, was used for analysis of the R28 rat retinal cell line. Total RNA was extracted from six million R28 cells (passage 48), followed by mRNA extraction with a Qiagen Oligotex Direct mRNA isolation kit. cDNA synthesis of intact mRNA was performed using dT-primed reverse transcriptase. Double-stranded cDNA was transcribed into cRNA by in vitro transcription. Biotinylated cRNA was hybridized overnight, washed six times, and stained with 6X-SSPE-T buffer (0.9 M NaCl, 60 mM NaH2PO4, 6 mM EDTA, and 0.005% Triton X-100) containing BSA and 1 mg/mL streptavidin-phycoerythrin as per the standard protocols of the Affymetrix Fluidics Station. DNA-Chip Analyzer (dChip) analysis was used to normalize the data and predict the presence or absence of mRNAs based on the average signal intensity of 11–24 spots for each gene and between replicate chips.
3. Dataset Description
The dataset associated with this Dataset Paper consists of 2 items which are described as follows.
Dataset Item 1 (Table). This item consists of 8799 rat genes and ESTs, along with gene descriptions, accession numbers, signal intensities, and “P” designating the gene present versus “A” designating the gene absent according to dChip analysis. dChip software was used to normalize the data and predict the presence or absence of a particular mRNA based on the average signal intensities among 11–24 spots for each gene and between replicate chips. The signal intensity values as well as the “P” and “A” columns are listed in duplicate for each row. We have included all of the genes in the table with both “A” and/or “P” designations based on signal intensity and dChip analysis. Genes that display both “A” and “P” were considered inconclusive. dChip analysis determined that of these 8799 genes and ESTs, 4131 were considered “present” in R28 cells. However, it is important to note that genes expressed at low levels or below our threshold levels may play a role in a variety of important biological processes.
- Column 1: Probe Set ID
- Column 2: Gene Title
- Column 3: Accession Number
- Column 4: Locus Link
- Column 5: Description
- Column 6: GS Control 1
- Column 7: GS Control 1 Call
- Column 8: GS Control 2
- Column 9: GS Control 2 Call
- Column 10: Average Signal
Dataset Item 2 (Table). A helpful updated annotation file with gene designations and probe set ID numbers based on tables freely available from the Affymetrix website, since the original microarray spreadsheet from our 2004 analysis was not annotated with gene symbols. This will aid the reader in finding a gene of interest based on gene symbols and gene products through the probe ID number.
- Column 1: Probe Set ID
- Column 2: Gene Title
- Column 3: Gene Symbol
4. Concluding Remarks
We present a full 8799 gene/EST dataset with designations of “present” or “absent” for the R28 retinal precursor cell line, which will make this dataset very useful for any investigator with an interest in using R28 cells for a particular molecular analysis. This complete dataset is not available as part of any other database or website; therefore, it is unique to this paper. This dataset cannot be considered absolute, but rather a starting point for further validation experiments. Ongoing and future studies on the behavior of R28 cells will be facilitated with this dataset in an open access format.
Dataset Availability
The dataset associated with this Dataset Paper is dedicated to the public domain using the CC0 waiver and is available at http://dx.doi.org/10.7167/2013/261063/dataset.
Disclosure
R28 cells and E1A-NR.3 cells were commercialized by KeraFAST in 2012. The corresponding author, G. M. Seigel, has a financial interest in the sale of these cell lines by KeraFAST (http://www.kerafast.com/c-167-retinal-cell-lines.aspx).
Acknowledgments
The authors thank Leighton Stein and Ram Varma of the Roswell Park Cancer Institute for microarray analysis. This study was supported by the NIH Grant P01 DC03600 (R. J. Salvi). G. M. Seigel is supported by the Cornell Center on the Microenvironment and Metastasis through Award no. U54CA143876 from the National Cancer Institute, R21CA127061, and NYSTEM C026412.
Dataset Files
- 261063.item.1.xlsx
Dataset Item 1 (Table). This item consists of 8799 rat genes and ESTs, along with gene descriptions, accession numbers, signal intensities, and “P” designating the gene present versus “A” designating the gene absent according to dChip analysis. dChip software was used to normalize the data and predict the presence or absence of a particular mRNA based on the average signal intensities among 11–24 spots for each gene and between replicate chips. The signal intensity values as well as the “P” and “A” columns are listed in duplicate for each row. We have included all of the genes in the table with both “A” and/or “P” designations based on signal intensity and dChip analysis. Genes that display both “A” and “P” were considered inconclusive. dChip analysis determined that of these 8799 genes and ESTs, 4131 were considered “present” in R28 cells. However, it is important to note that genes expressed at low levels or below our threshold levels may play a role in a variety of important biological processes.
- Column 1: Probe Set ID
- Column 2: Gene Title
- Column 3: Accession Number
- Column 4: Locus Link
- Column 5: Description
- Column 6: GS Control 1
- Column 7: GS Control 1 Call
- Column 8: GS Control 2
- Column 9: GS Control 2 Call
- Column 10: Average Signal
- 261063.item.2.xlsx
Dataset Item 2 (Table). A helpful updated annotation file with gene designations and probe set ID numbers based on tables freely available from the Affymetrix website, since the original microarray spreadsheet from our 2004 analysis was not annotated with gene symbols. This will aid the reader in finding a gene of interest based on gene symbols and gene products through the probe ID number.
- Column 1: Probe Set ID
- Column 2: Gene Title
- Column 3: Gene Symbol