or translocations affecting the 1p36 region have been described in other neuroblastoma patients [9,10]. Several other tumor types, such as breast cancer, malignant melanoma and colorectal cancer, are also characterized by frequent deletions or translocations of 1p36 [11]. In several types of tumors, including neuroblastoma, the shortest regions of overlap for 1p deletions partly coincide, indicating that the same TSGs may be implicated in different types of tumors [12]. A number of strong candidate TSGs were recently reported for 1p, including the CHD5 gene [13] and the microRNA-34a [14]. However, deletions in chromosome 1p36 are often very large in neuroblastoma [15], and it is likely that defects in more than one gene may contribute to the malignant phenotype. Expression profiling showed that expression levels of some genes located in the 1p35-36 region were decreased in neuroblastoma tumors and cell lines with 1p deletion as compared to 1p-normal samples [16,17]. Consequently, it was proposed that the underlying reason for the frequent deletion of this region in neuroblastoma is the decreased expression of several TSGs located in the 1p36 region and not the inactivation of one single classical TSG. Methods Probe generation We previously described a cosmid contig spanning the chromosome 17 breakpoint [18]. Nine regions lacking repetitive elements were chosen as probes for Southern analysis and amplified by PCR using PAC RPCI-1 880L8 as template. Probe 6, which is localized on the cosmid contig (GenBank Acc Nu AF148647) from position 36,914 to 37,391, was amplified with primers 59-TCCCAAAAGGCCAGTTTCACAC-39 and 59- TCTGCAGGCGTCTCATCTCAAC-39. Probe 9 is located from position 38,018 to 38,584, and was amplified with primers 59- GTTGTACCCCCTTGACTTCA-39 and 59-GTGCCCAG- CAGGAGATTCAAT-39. Southern blotting Genomic DNA from normal human placenta, the Chinese hamster ovary cell line A/3, and somatic cell hybrids 32-7A and 32-2F53VIII [7] was extracted using standard procedures. DNA was digested with BglII, HindIII, SacI, XhoI, XbaI, McoI, PvuII, KpnI or HincII. Blotting and hybridization were done according to standard procedures. GenomeWalker Libraries and PCR amplification GenomeWalker libraries were generated according to the manufacturer’s instructions (BD Biosciences, Palo Alto, CA, USA). For the 32-2F53VIII cell line, 2.5 mg of genomic DNA was digested with PvuII, DraI, StuI or EcoRV. For the 32-7A cell line, 2 mg of genomic DNA was digested with DraI, EcoRV, PvuII, ScaI or SspI. Subsequently, the appropriate GenomeWalker adaptors were ligated to the DNA. To amplify the segments overlapping the breakpoint, long- range PCR was done using Takara LA TAQ mix (Takara, Shiga, Japan) and gene-specific primers GSP1 (59-CCCCTCAGC- TCTGTGCATTTTGTCTA-39) and GSP2 (59-CCTCTTGCC- CCCACCTAGTGTTTATTT-39) for the 32-2F53VIII derived libraries, and primers GSP3 (59-CATAGTGGGGGACATCAT- GACAGTCAC-39) and GSP4 (59-ACACCACCAGCCTC- CCTCCATTTCTGA-39) for the 32-7A libraries. RACE analysis 39RACE was performed with a commercial kit according to the manufacturer’s instructions (Invitrogen, Merelbeke, Belgium). Neuroblastoma Breakpoint Briefly, 2 mg of total RNA isolated from the 32-7A cell line was reverse transcribed using the Adaptor Primer (AP). Two PCRs (PCR-A and PCR-B) were set up using the Unabridged Anchor Primer (UAP) and either GSP1 (59-GCCCTTATGACTCCAAC- CAG-39) or GSP2 (59-ATTGGCTCATCCTCTCATGTT-39). Nested PCRs were done using the Abridged Universal Anchor Primer (AUAP) and GSP2 (for nested PCR on PCR-A), or the AUAP and GSP3 (59-TCCCAGAAAATGAAAGTGATG-39) primer pair (nested PCR on PCR-B). To search for chimeric ACCN1 transcripts, we performed 59 and 39RACE analysis on cDNA from the 32-2F53VIII and 32-7A cell lines, respectively, using the Generacer kit according to the manufacturer’s instructions (Invitrogen). Briefly, 2 mg of total RNA was reverse transcribed using the supplied oligo-dT primer. In the 39RACE experiment, we performed a PCR with a GSP (59- AGCACTACAAACCCAAGCAGTTC-39) in combination with the supplied 39 adaptor primer. Nested PCR was done using a GSP (59-GTGGGCCATGACCTGAAGGATAT-39) in combina- tion with the supplied 39 nested primer. In the 59RACE experiment, we used a GSP (59-CTTGGATGAAAGGTGGCT- CAGACT-39) together with the supplied 59 adaptor primer. Nested PCR was performed using a GSP (59-TGATCTC- CAGCCCGTTGCCTGTC-39) and the supplied 59 nested primer. Products were cloned in pGEM-T Easy (Promega, Madison, WI, USA). mRNA expression profiling by real-time quantitative PCR For the analysis of the global NBPF expression pattern, we determined relative gene expression levels using an optimized two-step SYBR Green I RT-PCR assay employing several reference genes [19]. After establishment of amplification efficiencies of .95% for all primer pairs, the delta-Ct method was used for quantification. SYBR Green I core PCR reagents were obtained from Eurogentec (Seraing, Belgium) and used according to the manufacturer’s instructions. Reactions were run on an iCycler (Bio-Rad, California, USA). Gene expression levels were normalized using the geometric mean of four reference genes [19]. Relative expression levels for amplicons in the NBPF transcripts were measured in a panel of 31 neuroblastoma cell lines. For analysis of the NBPF1 expression pattern we determined the relative gene expression levels by an optimized two-step SYBR Green I RT-PCR assay using the two most stable housekeeping genes in neuroblastoma cell lines [19]. One microgram of total RNA was treated with the RQ1 RNase-free DNase according to the manufacturer’s instructions (Promega). Before synthesizing cDNA, treated RNA samples were desalted on Microcon-100 spin columns (Milipore, Bedford, USA). cDNA was synthesized at 54uC using gene-specific primers (NBPF1: 59- CTGGGTGGACTTCGCGTAACTT-39, GAPD: 59-GGCATG- GACTGTGGTCATGAG-39 and HPRT1: 59-GGTCCTTTTC- ACCAGCAAGCT-39) and Superscript III reverse transcriptase according to the manufacturer’s instructions (Invitrogen). RT- PCR mixes contained 20 ng template cDNA, LightCycler 480 SYBR Green I Mastermix (Roche Diagnostics GmbH, Man- nheim, Germany) and 900 nM forward and reverse primers. Reactions were performed on a LightCycler 480 (Roche Diagnostics) using the following protocol: incubation at 95uC for 5 min, then 50 cycles at 95uC for 10 sec, 66uC for 30 sec, and 72uC for 1 sec. Primer sequences for the reference genes are deposited in RTPrimerDB, a public database for real-time PCR primers (http://medgen.ugent.be/rtprimerdb/; [20]): UBC, SDHA, HPRT1, and GAPD. The primer sequences for the NBPF PLoS ONE | www.plosone.org 2 May 2008 | Volume 3 | Issue 5 | e2207