Primary human neuroblastoma Total RNA was extracted from each frozen tissue according to the AGPC method (Chomczynski and Sacchi, 1987). As a reference, a mixture of equal amounts of RNA from each of four neuroblastoma cell lines (NB69, NBL-S, SK-N-AS, and SH-SY5Y) were used. Ten micrograms each of total RNA were labeled with the CyScribe RNA labeling kit according to the manufacturer's manual (Amersham Pharmacia Biotech), followed by probe purification with the Qiagen MinElute PCR purification kit (Qiagen). RNAs extracted from primary neuroblastoma tissues and RNAs of the reference mixture were labeled with Cy3- and Cy5- conjugated deoxynucleotides (Amersham), respectively, and were used as probes in 3.4 x SSC / 0.3% SDS together with 40 micrograms yeast tRNA and 100 micrograms polyA for suppression. Prior to hybridization, each microarray was soaked at room temperature for 30 min in Blocking solution containing 3% BSA, 0.2M NaCl, 0.1M Tris/HCl (pH 7.5) and 0.05% Triton X-100, dried at 37 degree celcius for 15 min, and washed with TE twice. After removing the remaining TE by centrifugation at 1000 rpm for 2 min, 20 microlitter of probe was applied onto the microarray and covered with a cover glass. The microarray was transferred to humidiated tightly covered box and incubated in 65 degree celcius chamber for 16 hours. The slides were washed twice for 5 min with 2xSSC, 0.1% SDS and then twice with 0.2xSSC, 0.1% SDS for 5 min at room temperature followed by centrifugation at 1000 rpm for 2 min. Hybridized microarrays were scanned using the Agilent G2505A confocal laser scanner (Agilent Technologies, Inc.), and fluorescent intensities were quantified using the GenePix Pro microarray analysis software (Axon Instruments, Inc.). Keywords = Primary human neuroblastoma
