Comparison of expression profile between human Müller cells, HMCL-I and HMCL-II

Müller cells are the principal glial cells in the retina. Alterations in Müller cell behaviour are observed in retinal tissue from patients with proliferative diabetic retinopathy. The purpose of this study was to compare gene and protein expression profile of normal human Müller cells (NHMC) with two spontaneously human Müller cell lines generated from type 1 (HMCL-I) and type 2 (HMCL-II) diabetic donors using Serial Analysis Gene Expression (SAGE). Approximatively 50 000 tags were sequenced for each of the three SAGE libraries. Identification of the transcripts was obtained by matching the 15bp (CATG + 11bp) with the UniGene and GenBank databases. Classification of the genes was based upon the updated information of the genome directory found at the NCBI website. SAGE allowed us to characterize the entire transcriptome of human Müller cells and to compare these data with those from Müller cells of type 1 and 2 diabetic donors. Keywords: Müller cells, SAGE, HMCL-I, HMCL-II, expression profile comparison Polyadenylated RNA was extracted with the Oligotex mRNA Purification System (Qiagen), annealed with the biotin-5 -T18-3 primer and converted to cDNA with the cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). The resulting cDNA library was digested with NlaIII (anchoring enzyme), and the 3’ restriction fragments were isolated with streptavidin-coated magnetic beads (Dynal Biotech, Oslo, Norway) and separated into two populations. Each population was ligated to one of the two annealed linker pairs and extensively washed to remove unligated linkers. The tag beside the most 3’ NlaIII restriction site (CATG) of each transcript was released by digestion with BsmFI (tagging enzyme). The blunting kit from Takara Co. (Otsu, Japan) was used for the blunting and ligation of the two tag populations. The resulting ligation products containing the ditags were amplified by PCR with an initial denaturation step of 1 min at 950C, followed by 22 cycles of 20 s at 940C, 20 s at 600C and 2 s at 720C with 27 bp primers. The PCR product was digested with NlaIII, and the band containing the ditags was extracted from the acrylamide gel. The purified ditags were self-ligated to form concatemers. The concatemers of 500 to 1800bp were isolated by agarose gel. The resulting DNA fragments were ligated into the SphI site of pUC19 and cloned into UltraMAX DH5-alpha-FT (Invitrogen). White colonies were screened by PCR to select long inserts for automated sequencing. Sequence files were analyzed by the SAGEana program, a modification of SAGEparser (ftp://ftp.pbrc.edu/public/eesnyder/SAGE/). Tags corresponding to linker sequences were discarded, and duplicate concatemers were counted only once. We used the comparative count display (CCD) test to identify the transcripts that were differentially expressed significantly (P<0.05) between the groups with more than a twofold change. The CCD test makes a key-by-key comparison of two key-count distributions by generating a probability that the frequency of any key in the distribution differs by more than a given fold factor from the other distribution.