Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets-2

Decorin) (), () and () of the RGP library; (14-3-3 ξ) () identified in both libraries; and () and () from the Met library were isolated and used as probes for hybridization in Northern blots containing total RNA from the melanoma cell lines indicated above the panels – mean that the corresponding cell line was not included in the Northern blot, and were introduced to allow alignment among panels. Northern blots were prepared as described in Figure 1. () identified in the Met library was validated by RT-PCR. For RT-PCR, total RNA samples (2 μg) from the indicated cell lines were, after DNase treatment, submitted to reverse transcription with Superscript II (Invitrogen) using oligo dT as primer and the cDNA was used as template for PCR amplification with primers. After 25, 28, 30 and 32 amplification cycles, 5 μl aliquots were collected for agarose gel electrophoresis. As endogenous control, a pair of primers for the (β-actin) mRNA was used. : Control RT-PCR amplification using as template RNA (DNase treated) without prior reverse transcription.<p><b>Copyright information:</b></p><p>Taken from "Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets"</p><p>http://www.biomedcentral.com/1471-2407/8/19</p><p>BMC Cancer 2008;8():19-19.</p><p>Published online 22 Jan 2008</p><p>PMCID:PMC2267200.</p><p></p

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets-4

E expression data for each gene group were submitted to SAM (FDR = 0) in a two-class analysis for detection of genes differentially expressed between primary and metastatic tumors and between non-neoplastic (skin and melanocytic nevi) and neoplastic (primary and metastatic melanomas) samples. The results from SAM analysis were extracted using SAMTERS and visualized by CLUSTER 3.0 and Java TreeView – Red and green squares represent genes up-regulated and down-regulated, respectively. (A) Expression profiles from primary and metastatic tumors for genes from the RGP library. (B – C) Expression profiles from non-neoplastic and neoplastic samples for genes from the RGP (B) and Met (C) libraries. Vertical blue lines on the left side indicate: (B) Genes from the RGP library that showed up-regulation in non-neoplastic samples; and (C) Genes from the Met library up-regulated in neoplastic tissues.<p><b>Copyright information:</b></p><p>Taken from "Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets"</p><p>http://www.biomedcentral.com/1471-2407/8/19</p><p>BMC Cancer 2008;8():19-19.</p><p>Published online 22 Jan 2008</p><p>PMCID:PMC2267200.</p><p></p

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets-6

duplicate samples of subtracted (S1 and S2) or non-subtracted (NS) cDNA of the RGP (WM1552C) and the metastatic (a pool of WM9, WM852, 1205Lu and WM1617) cell lines. PCR-2 represents the PCR product generated after 10 amplification cycles by nested-PCR using a specific primer for each adaptor. Note the difference between the subtracted and non-subtracted profiles.<p><b>Copyright information:</b></p><p>Taken from "Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets"</p><p>http://www.biomedcentral.com/1471-2407/8/19</p><p>BMC Cancer 2008;8():19-19.</p><p>Published online 22 Jan 2008</p><p>PMCID:PMC2267200.</p><p></p

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets-1

duplicate samples of subtracted (S1 and S2) or non-subtracted (NS) cDNA of the RGP (WM1552C) and the metastatic (a pool of WM9, WM852, 1205Lu and WM1617) cell lines. PCR-2 represents the PCR product generated after 10 amplification cycles by nested-PCR using a specific primer for each adaptor. Note the difference between the subtracted and non-subtracted profiles.<p><b>Copyright information:</b></p><p>Taken from "Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets"</p><p>http://www.biomedcentral.com/1471-2407/8/19</p><p>BMC Cancer 2008;8():19-19.</p><p>Published online 22 Jan 2008</p><p>PMCID:PMC2267200.</p><p></p

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets-5

Stages of tumor progression supported the selection of WM1552C cell line as the RGP representative for suppression subtractive hybridization against a pool of metastatic cell lines. Samples of 20 μg of total RNA from different melanoma cell lines were submitted to electrophoresis in 1% agarose-formaldehyde gel and transferred to nylon membrane (Hybond N, Amersham Pharmacia Biotech) by standard methods. Fragments of the indicated genes were radiolabeled with [α-32P]-dCTP by random-priming (Rad-prime kit, Invitrogen) and used as probes for Northern blot hybridization. In order to correct for loading differences, after stripping, the blots were probed with a ACTB (β-actin) cDNA fragment.<p><b>Copyright information:</b></p><p>Taken from "Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets"</p><p>http://www.biomedcentral.com/1471-2407/8/19</p><p>BMC Cancer 2008;8():19-19.</p><p>Published online 22 Jan 2008</p><p>PMCID:PMC2267200.</p><p></p

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets-3

total number of genes per human chromosome for each library; (B and C) Represent the chromosome locations for all genes identified in the RGP (B) and Met (C) libraries, along the length (bp) of all human chromosomes. The absence of genes mapping to Y chromosome in the RGP library is not explained by lack of this chromosome since the RGP cell line WM1552C was obtained from a male patient.<p><b>Copyright information:</b></p><p>Taken from "Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets"</p><p>http://www.biomedcentral.com/1471-2407/8/19</p><p>BMC Cancer 2008;8():19-19.</p><p>Published online 22 Jan 2008</p><p>PMCID:PMC2267200.</p><p></p

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets-0

Stages of tumor progression supported the selection of WM1552C cell line as the RGP representative for suppression subtractive hybridization against a pool of metastatic cell lines. Samples of 20 μg of total RNA from different melanoma cell lines were submitted to electrophoresis in 1% agarose-formaldehyde gel and transferred to nylon membrane (Hybond N, Amersham Pharmacia Biotech) by standard methods. Fragments of the indicated genes were radiolabeled with [α-32P]-dCTP by random-priming (Rad-prime kit, Invitrogen) and used as probes for Northern blot hybridization. In order to correct for loading differences, after stripping, the blots were probed with a ACTB (β-actin) cDNA fragment.<p><b>Copyright information:</b></p><p>Taken from "Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets"</p><p>http://www.biomedcentral.com/1471-2407/8/19</p><p>BMC Cancer 2008;8():19-19.</p><p>Published online 22 Jan 2008</p><p>PMCID:PMC2267200.</p><p></p

Identification of unannotated exons of low abundance transcripts in and cloning of a new serine protease gene upregulated upon injury-1

<p><b>Copyright information:</b></p><p>Taken from "Identification of unannotated exons of low abundance transcripts in and cloning of a new serine protease gene upregulated upon injury"</p><p>http://www.biomedcentral.com/1471-2164/8/249</p><p>BMC Genomics 2007;8():249-249.</p><p>Published online 24 Jul 2007</p><p>PMCID:PMC1949825.</p><p></p>re hybridized to the probes indicated at the bottom of each blot. The estimated size (kb) of each transcript is indicated on the left

Identification of unannotated exons of low abundance transcripts in and cloning of a new serine protease gene upregulated upon injury-3

<p><b>Copyright information:</b></p><p>Taken from "Identification of unannotated exons of low abundance transcripts in and cloning of a new serine protease gene upregulated upon injury"</p><p>http://www.biomedcentral.com/1471-2164/8/249</p><p>BMC Genomics 2007;8():249-249.</p><p>Published online 24 Jul 2007</p><p>PMCID:PMC1949825.</p><p></p> . These sequences are from proteins that presented the highest similarity with SP212 catalytic domain sequence (e-value < -20) in searches against the NCBI databank, using Blastx. The alignment was performed using Clustalw (20). SP55, SPH144 and SP60, as well SP212, localize in the same region (88A12-B1) of chromosome 2R, whereas SP186 is located in chromosome X

Identification of unannotated exons of low abundance transcripts in and cloning of a new serine protease gene upregulated upon injury-0

<p><b>Copyright information:</b></p><p>Taken from "Identification of unannotated exons of low abundance transcripts in and cloning of a new serine protease gene upregulated upon injury"</p><p>http://www.biomedcentral.com/1471-2164/8/249</p><p>BMC Genomics 2007;8():249-249.</p><p>Published online 24 Jul 2007</p><p>PMCID:PMC1949825.</p><p></p>ch [16]