title: CAGE Lab - Head and Neck carcinoma cDNA microarray
status: Public on Jun 22 2009
contact.city: São Paulo
contact.first: Eduardo
contact.middle: Moraes
contact.last: Reis
contact.email: emreis@iq.usp.br
contact.state: SP
contact.address: Av. Prof. Lineu Prestes, 748
contact.department: Biochemistry
contact.country: Brazil
contact.institute: University of São Paulo
contact.phone: +55-11-30912173
organism: Homo sapiens
manufacture_protocol: We selected 136 genes previously identified by the Head and Neck Annotation Consortium (Reis et al., 2005, Cancer Research 65:1693) to be spotted in the cDNA microarray. The criteria for selection of the genes were based on known genome mapping information. Thus, genes that map to loss or gain regions involved in head and neck carcinogenesis, according to Knuutila et al. (Knuutila et al., 2000, Am J Pathol 157:689) were selected.
  Complementary DNA of the Hep2 (Human laryngeal carcinoma) cell line served as template for the amplification of probes from microarray spotting. Gene-specific primers for PCR were designed using the Primer 3.0 program.
  In brief, for the PCR, 1 uL cDNA was mixed with 2.5 uL 10x buffer, 1 uL PCR primer (10 pmol/L), 4 uL dNTPs (1.25 mM), 0.75 uL MgCl2 (50 mM) and 0.2 uL Taq DNA Platinum (Invitrogen). PCR conditions were: 94ºC for 4 min, followed by 35 cycles at 94ºC for 30 s, 60ºC for 1 min and 72ºC for 1 min.
  The products obtained by PCR had the expected sizes and have average length of 600 bp. From all products, 5uL were fractionated by electrophoresis through a 1% agarose gel for size verification, and the remaining 95uL were purified by filtration on Multiscreen plates (Millipore cat. # MAFB NOB 50). cDNA samples were diluted 1:1 in DMSO and spotted onto silane-coated, reflective Type-7 glass slides (GE Healhcare), using a Generation III microarray spotter robot (GE Healhcare). Twelve replicates of 4 different plant/bacterial DNA fragments with no similarity to human sequences were evenly distributed along the slide to serve as negative controls of nonspecific hybridization (Lucidea ScoreCard − GE Healthcare). Following spotting, cDNAs were fixed to the slide surface by UV cross-linking (500 mJ) and kept in a low-humidity environment until use.
  Based on an informatics analysis of the HN transcriptome to identify candidates for LOH in Head and Neck tumors, an additional set of 196 cDNA clones were included in the array, comprising partial sequences of protein-coding transcripts (123 sequences), as well as of transcripts mapping to intronic (53 sequences) or intergenic regions of the genome (20 sequences). The original sequencing clone collection was stored frozen in bar-coded 96-well plates, and the selected clones were re-arrayed in an automated robotic operation. cDNA fragments were generated from these clones by PCR amplification with universal primers, purified by filtration and spotted as described above. Each probes was deposited 3-4 times, in duplicate, in the arrays. Thus, 6 to 8 replicates of each probe are spotted in the microarrays.