Diagnosis of the Small Round Blue Cell Tumors Using Multiplex Polymerase Chain Reaction

Abstract

The small round blue cell tumors of childhood, which include neuroblastoma, rhabdomyosarcoma, nonHodgkin's lymphoma, and the Ewing's family of tumors, are so called because of their similar appearance on routine histology. Using cDNA microarray gene expression profiles and artificial neural networks (ANNs), we previously identified 93 genes capable of diagnosing these cancers. Using a subset of these, together with some additional genes (total 39), we developed a multiplex polymerase chain reaction (PCR) assay to diagnose these cancer types. Blinded testing of 96 new samples (26 Ewing's family of tumors, 29 rhabdomyosarcomas, 24 neuroblastomas, and 17 lymphomas) using ANNs in a complete leaveone-out analysis demonstrated that all except one sample were accurately diagnosed as their respective category. Moreover, using an ANN-based gene minimization strategy in a separate analysis, we found that the top 31 genes could correctly diagnose all 96 tumors. Our results suggest that this molecular test based on a multiplex PCR reaction may assist the physician in the rapid confirmation of the diagnosis of these cancers. (J Mol Diagn 2007, 9

Upregulation of Poly (ADP-Ribose) Polymerase-1 (PARP1) in Triple-Negative Breast Cancer and Other Primary Human Tumor Types

“…41 Real-time PCR-based transcriptional profiling and multiplex assays used for this study were designed to detect RNA from FFPE samples or from frozen tissues. RNA quality and quantity were analyzed using the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA) and the RiboGreen RNA Quantitation Kit (Invitrogen, Carlsbad, CA) as recommended by the manufacturer.…”

“…Reverse transcription (RT) reactions were carried out as previously described. 41 Briefly, 25 ng of RNA was converted into single-stranded cDNA, according to the XP Start kit (Beckman Coulter) protocol. The reactions were incubated for 1 minute at 48°C, 5 minutes at 37°C, 60 minutes at 42°C, and then 5 minutes at 95°C.…”

“…QRT-PCR-based transcriptional profiling was then performed on each sample as previously described. 41 Samples were analyzed with a GeXP Genetic Analysis System (Althea Technology, AltheaDx) and eXpress Analysis module of the GeXP Genetic Analysis System. 41 The expression of each target gene was analyzed relative to the expression of β-glucuronidase (GUSB) within the same reaction.…”

“…41 Samples were analyzed with a GeXP Genetic Analysis System (Althea Technology, AltheaDx) and eXpress Analysis module of the GeXP Genetic Analysis System. 41 The expression of each target gene was analyzed relative to the expression of β-glucuronidase (GUSB) within the same reaction. Data were reported as the mean and standard deviation of 3 independent assessments for each sample.…”

Involvement of the MADS-Box GeneZMM4in Floral Induction and Inflorescence Development in Maize

“…Multiplex gene expression analysis was carried out using the GenomeLab GeXP analysis system (Beckman Coulter) as described previously (Chen et al, 2007). The gene-specific sequences of the chimeric primers were as follows: ZMM4 forward 5#-GGCGAAGGTCGAGACAATAC-3# and reverse 5#-GCT-GGCTCTTCCTTGTTCTG-3#, ZMM15 forward 5#-GCTAGCTGTGACGTTAT-GCT-3# and reverse 5#-CCTCTCCGCTAGGACAACCT-3#, ZMM24 forward 5#-GCTCCTGTGTGCAGTGTGTC-3# and reverse 5#-GCGCTATCAGCGAC-CTCTT-3#, ZMM31 forward 5#-AGCATATAGCCCAGCACCAC-3# and reverse 5#-CCCTGGGTTTTTTCGAGCT-3#, and a-tubulin forward 5#-GTT-CAATGCTGTTGGTGGTG-3# and reverse 5#-GTCCAGGAGGACTGCAA-CAT-3#.…”