BMC Cancer 2008, 8:19 http://www.biomedcentral.com/1471-2407/8/19 FViagliudarteio3n by Northern blot and RT-PCR of the expression pattern of seven genes identified in the SSH libraries Validation by Northern blot and RT-PCR of the expression pattern of seven genes identified in the SSH librar- ies. Frames depict the names of cell lines used in the construction of the libraries. The inserts of cDNA clones corresponding to the genes DCN (decorin) (A), ALS2CR7 (B) and MBOAT1 (C) of the RGP library; YWHAZ (14-3-3 ξ) (D) identified in both libraries; and MITF (E) and PLP1 (F) 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 – Blank lanes mean that the corresponding cell line was not included in the Northern blot, and were introduced to allow alignment among panels. Northern blots were pre- pared as described in Figure 1. HLA-DRA (G) 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 HLA-DRA 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 ACTB (β-actin) mRNA was used. C: Control RT-PCR amplification using as template RNA (DNase treated) without prior reverse transcription. Page 8 of 18 (page number not for citation purposes)