D818 Nucleic Acids Research, 2009, Vol. 37, Database issue CTdatabase is mRNA expression data. These data are divided between ‘High-throughput’ (obtained using large-scale techniques), ‘Tested by Ludwig Institute for Cancer Research’ (RT-PCR) and ‘Published literature’ (manual annotation). HIGH-THROUGHPUT DATA Three diﬀerent sources of data were utilized: Serial Analysis of Gene Expression [SAGE, (12)], Massive Parallel Signature Sequencing [MPSS, (13)] and Expressed Sequence Tags [EST, (14)]. ESTs are cDNA fragments of a hundred or more nucleotides (nt). SAGE data are comprised of ‘short’ or ‘long’ sequence tags, 10 nt and 17 nt, respectively, from the 30-end of mRNAs. Massive Parallel Signature Sequencing tags are 13 nt tags obtained using an alternative sequenc- ing protocol. In all cases, the number of EST, SAGE or MPSS tags reﬂect the number of mRNA copies in a cell; the higher the number of tags observed for a given gene, the higher the expression of the gene. The CTdatabase contains a heat map of color-coded expression levels of CT antigens. Genes not conﬁrmed as CT antigens have their expression levels presented in the heat map, but are ﬂagged as not being testis restricted. SAGE AND MPSS Both SAGE and MPSS tags are computationally pre- dicted for each CT gene (mRNA RefSeq) using custom programs that simulate the SAGE/MPSS protocols. To do this, we located the 30 most CATG site (the enzyme cleav- age site used for SAGE) or GATC (the cleavage site used for MPSS) and extracted the putative downstream tag. To guarantee that the tag is derived from the 30-end of a given CT antigen only mRNAs with a poly-A tail (>5 As) are used. When a given tag is observed in more than one gene, it is not accepted as a bona-ﬁde tag. When the tag belongs to gene copies, a warning is displayed, cautioning the user that the expression level may not be correctly reported. The frequency of each predicted tag (expression level) in diﬀerent tissues was downloaded from SAGE Genie (15) at the Ludwig Institute for Cancer Research (LICR) FTP site (ftp://ftp.licr.org/pub/databases/trome/human/) and parsed to generate a heat map. Only tissues with more than 100 000 tags are shown. Library annotation (normal/cancer, sample source, etc) were downloaded from SAGE Genie. A limited number of corrections were manually performed. ESTs The number of ESTs per gene contained in UniGene clus- ters with at least 60 000 sequences were normalized per million and also presented as a heat map. Tissue and health state annotation provided by UniGene were used to separate the cancer libraries presented. Normalized and subtracted libraries were excluded from the data to avoid sampling biases. Intronless ESTs were excluded to avoid bias from genomic DNA contaminations. Some CT antigen genes had more than one correspond- ing UniGene cluster due to gene copies. For these cases, UniGene clusters were merged following manual inspec- tion and a corresponding warning is displayed in the entry. RT-PCR In addition to third-party expression data, the CTdatabase provides an RT-PCR analysis of the expres- sion levels of all CT antigen genes. A standardized analysis was undertaken in the same set of cDNA preparations from normal human tissues as well as selected human cancer cell lines. Thus the expression of 106 genes was analyzed in a panel of 22 normal tissues and 34 cancer cell lines by RT-PCR at the LICR New York Branch. Gel images are displayed and the experimental conditions, including primer sequences, PCR cycles and temperatures are provided. LITERATURE DATA Manually curated information retrieved from the litera- ture is included in the CTdatabase. A list of normal tissues expressing the referred CT, as indicated by literature refer- ences, is shown. Data on expression of individual CT anti- gens in neoplasias were annotated and are presented according to tumor type and subtype, indicating the level of expression. A list of cell lines expressing each CT gene is also presented. For all literature information, the experimental method is provided as well as links to the PubMed references. THE ‘PROTEIN EXPRESSION’ TAB The protein expression tab includes manually reviewed information from the literature on CT protein expression in normal tissues, tumor tissues and tumor cell lines. The methodologies employed in the experiments and the PubMed references are provided. A list of antibodies raised against CT antigens as published in the literature is included. THE ‘IMMUNE RESPONSE’ AND ‘PubMed’ TABS The ‘Immune response’ tab is divided into three sections containing information manually curated from the litera- ture respectively focused on ‘Humoral immune response’, ‘Cellular immune response’ and ‘Induced immune response’. The ﬁrst section contains data on spontaneous humoral immune responses to CT antigens in patients with diﬀerent tumor types, including the frequency of patients with antibodies against the antigen where avail- able. The technique used for detection of the antibodies and the PubMed article reference is shown for each entry. The ‘Cellular immune response’ section contains informa- tion on spontaneous cellular immune responses against the CT antigen in cancer patients. This tab also displays