BMC Cancer 2008, 8:19 http://www.biomedcentral.com/1471-2407/8/19 the blot and by UV cross-linking. Pre-hybridization was done in a solution containing 7% SDS, 1% BSA, 1 mM EDTA, and 0.5 M NaHPO4 pH 7.5 [36], at 65°C for at least 1 h, in a 30/3,5 cm roller bottle in a hybridization oven. For probe generation, digested inserts were gel purified (Qiaex II kit-Qiagen, Valencia, CA) and about 50 ng were radio-labeled with [α-32P]-dCTP by random-priming (Rad-prime kit, Invitrogen, Carlsbad, CA, USA). Unincor- porated nucleotides were removed by gel filtration through a G-50 Sephadex column. The hybridization was performed for 18 h using the probe to 1 × 106 CPM/ml hybridization solution. The blots were washed in the fol- lowing manner: one time in 2 × SSC, 0,2% SDS, for 5 min, at room temperature; two times in 1 × SSC, 0,2% SDS, for 30 min each, at 65°C; and one time in 0,2 × SSC, 0,2% SDS, for 30 min, at 65°C. Then, the blots were covered in clear plastic wrap and exposed to a Phosphoimager screen (Molecular Dynamics, Piscataway, NJ, USA). In order to correct for different lane loads, blots were stripped at 100°C in 0.5% SDS and probed with a fragment for ACTB (β actin) gene. RT-PCR for HLA-DRA gene For RT-PCR, total RNA was treated with DNase I (Promega, Madison, WI, USA) at 1 U/2 μg of total RNA in 10 μL reaction volume and incubated for 30 min at 37°C, followed by enzyme inactivation by addition of 1 μL of 20 mM EDTA and incubation for 15 min, at 65°C. cDNA synthesis was performed using 2 μg of total RNA in 20 μL reaction with Superscript II Reverse Transcriptase (Invitro- gen, Carlsbad, CA, USA), according to the manufacture's instructions, using 4 μL of 5× first-strand buffer, 1 μL of 10 mM dNTP, 200 U Superscript II enzyme, 2 μL of 0.1 M DTT, and 250 ng oligo dT primer (Invitrogen, Carlsbad, CA, USA). For PCR reactions, 1 μL of each synthesized cDNA was used as template in a reaction volume of 50 μL containing 200 μM dNTPs, 1,5 mM MgCl2, 0.25 μM each primer, and 1 U Taq DNA polymerase in the manufac- ture's recommended buffer (Invitrogen, Carlsbad, CA, USA). The reaction was allowed to denature for 4 min at 94°C, followed by amplification (25, 28, 30 e 32 cycles: 45 s at 94°C, 1 min at 55°C, 1 min at 72°C). At indicated cycles, a 5 μL sample was colleted from each reaction. Amplification of ACTB (β actin) cDNA was done as con- trol for mRNA content. The following forward (F) and reverse (R) primers were used: F-ACAGAGCGCCCAA- GAAGAAAA and R-CTCAAAGCTGGCAAATCGTC for amplification of HLA-DRA; and F-GGCATCGTGAT- GGACTCCG and R-GGAAGGTGGACAGCGA for ACTB. PCR products were loaded onto a 1% agarose gel and elec- trophoresed in TAE buffer. Gels were subjected to ethid- ium bromide staining and were imaged in a UV transilluminator using a digital Kodak camera. Analysis of the expression profile of genes represented by subtractive clones in a publicly available microarray study of melanoma samples We downloaded from the PNAS website [37] the table number 10 containing the normalized and log2 trans- formed expression data from the microarray study described by Haqq et al [16]. This table presents the data of the comparison of expression profiles of samples from normal skin, nevi, primary and metastatic melanomas using a microarray from Research Genetics containing 20,862 human cDNA clones. Using a locally developed computer script, we extracted the expression data from their microarray analysis for all genes that were also repre- sented in both of our subtractive libraries (RGP and Met). The expression values of both groups of genes were sub- mitted to SAM (Significance Analysis of Microarrays) [38] software in a two class comparison, first to detect the genes presenting differential expression between primary and metastatic melanomas and then, in a extended analy- sis, between non-neoplastic tissues (skin and nevi) and tumors (primary and metastatic melanomas). The results of SAM were extracted using the software SAMSTER [39], submitted to hierarchical clustering using CLUSTER and then visualized by JAVATREEVIEW [40]. Results Selection of cell lines for generation of two cDNA SSH libraries and sequencing analysis We used the SSH approach to identify populations of mRNA that distinguish between a non-tumorigenic RGP cell line (WM1552C) and a pool of four metastatic cell lines (WM9, WM852, WM1617, 1205Lu). In order to reduce individual genetic variations, we initially aimed to use a pool of less aggressive cell lines as we do for the met- astatic cells. However, as shown here based on several cri- teria we failed to find more than one among six RGP/VGP cell lines tested to fit in a "less aggressive" phenotype. Also, we were discouraged to include a VGP cell line in the study based on the rationale that cells from VGP tumors are more heterogeneous, as pointed out in the Back- ground section. For selecting the cell lines, we checked on the expression of three known molecular markers of melanoma progres- sion, KISS1 and RHOC mRNAs and the αvβ3 integrin, in the panel of melanoma cell lines used here. KISS1 mRNA expression was detected by Northern blot only in the RGP cell line WM1552C (Fig. 1A). So even the other two RGP cell lines (WM35 and WM1789) included in our study failed to show detectable levels of KISS1 mRNA, although by using a more sensitive method (RT-PCR/Southern blot- ting), a weak expression of KISS1 transcript in the WM35 cell line in contrast with lack of expression in WM793 (VGP) and 1205Lu (metastatic) was previously reported [21]. Page 4 of 18 (page number not for citation purposes)