BMC Cancer 2008, 8:19 http://www.biomedcentral.com/1471-2407/8/19 detection of low copy transcripts and, in contrast to microarray analysis, it allows the identification of unknown genes or non-coding RNAs, thus representing an alternative and complementary approach for differen- tial expression analyses (for a comparative study see [30]). In the present work, we used SSH approach to compare a non-tumorigenic cell line whose behavior and expression profile of a particular set of genes are suggestive of low aggressiveness to a pool of established, highly tumori- genic and metastatic cell lines, aiming to identify sets of genes potentially involved in maintaining low versus high aggressiveness status in melanoma. Methods Cell culture The cell lines used in this work represent the three major stages of melanoma progression: radial growth phase (WM35, WM1552C and WM1789), vertical growth phase (WM278, WM793 and WM902) and metastasis (WM9, WM852, 1205Lu and WM1617). All melanoma cell lines were kindly provided by Dr. Meenhard Herlyn (Wistar Institute, Philadelphia, PA). The cells were maintained in melanoma medium, consisting of four parts of MCDB153 (Sigma, Saint Louis, MO, USA) and one part of L-15 (Inv- itrogen, Carlsbard, CA, USA), supplemented with 2 mM CaCl2, 5 μg/ml insulin and 2% fetal bovine serum (Invit- rogen, Carlsbard, CA, USA). Isolation of RNA and mRNA Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbard, CA, USA) and mRNA was isolated from total RNA using the Oligotex™ mRNA kit (Qiagen, Valencia, CA, USA) according to manufacturer's instructions. The integrity of RNA and mRNA was checked on a 1% formal- dehyde agarose gel. Suppression subtractive hybridization (SSH) The subtractive libraries were constructed using the Clon- tech PCR-Select™ cDNA Subtraction kit (Clontech, Palo Alto, CA, USA). Briefly, 1 μg of mRNA (poly dA+ RNA) from WM1552C (RGP-like) and equal amount from a pool of four metastatic cell lines (WM9, WM852, 1205Lu and WM1617) were used for double strand cDNA synthe- sis, and the resulting cDNA was digested with Rsa I. For the RGP-library, the digested cDNA from WM1552C (as a Tester) was split into two groups and linked to either adaptor I or adaptor 2R. Subtractive hybridization was performed by annealing an excess of the metastatic cell cDNA (as a Driver) with each sample of adaptor-ligated tester cDNA. The cDNAs were heat denatured and incu- bated at 68°C for 8 hours (h). After the first hybridization, the two samples were mixed together and hybridized again with freshly denatured driver cDNA for 20 h at 68°C. The two rounds of hybridization would generate a normalized population of tester specific cDNAs with dif- ferent adaptors on each end. After filling in the ends, two rounds of PCR amplification were performed to enrich for the desired cDNAs containing both adaptors. The opti- mized cycling for the first and second PCR rounds, to increase representation and reduce redundancy of sub- tracted cDNAs, were 27 and 10 cycles, respectively. The Met-library was constructed using the same approach but with cDNA from the metastatic cell lines as a Tester and cDNA from WM1552C as a Driver. Cloning of the subtracted cDNAs The amplified products containing the subtracted cDNAs from both subtraction processes (4 μL) were independ- ently ligated into a pGEM-Teasy vector (Promega Co., USA) and transformed into E. coli strain DH5α. Bacteria were supplied with 800 μL of SOB medium, incubated for 1 h at 37°C, and subsequently plated onto agar plates containing 100 μg/mL ampicillin, 100 mM IPTG and 100 mg/mL X-gal at 37°C, for 20 h. White colonies were inoc- ulated into 96-well plates containing 150 μL of 2× YT liq- uid medium supplemented with 100 μg/mL ampicillin. The cultures were grown overnight, without shaking, at 37°C. PCR amplification to check for the positive clones, i.e., to confirm the presence of insert, and to generate sequencing templates was performed as previously described [31]. Sequencing, annotation and sequence analysis A total of 753 clones from both libraries were sequenced using the kit DYEnamic ET dye terminator cycle sequenc- ing (Amersham-Pharmacia, Pollards Wood, UK) and a M13 primer in the capillary DNA sequencer Megabace 1000 (Amersham-Pharmacia Biotech, Pollards Wood, UK). The BLAST program was used to search for the cDNA sequence similarity of isolated clones in the GenBank [32]. Annotated sequences were submitted to functional annotation according to the Gene Ontology database, using the tool GOTM-Gene Ontology Tree Machine [33]. For the chromosome distribution analysis, chromosome locations of all genes/ESTs were obtained from GenBank accession number reports or through BLAT alignment [34], and then the total number of genes per human chro- mosome for each library was plotted in a bar graphic. Graphics showing the gene distribution along each human chromosome was generated using the "Chromo- somal Distribution Chart" tool from the WebGestalt home page [35]. Northern blot For Northern blot preparation, 20 μg of total RNA was separated by 1% formaldehyde-agarose gel electrophore- sis and transferred to nylon membrane (Hybond N, Amer- sham Pharmacia Biotech, Pollards Wood, UK) by standard methods. RNA was fixed to membrane by baking Page 3 of 18 (page number not for citation purposes)