No abstract

Background: DNA microarray technology has permitted the analysis of global gene expression profiles for several diseases, including cancer. However, standard hybridisation and detection protocols require micrograms of mRNA for microarray analysis, limiting broader application of this technology to small excisional biopsies, needle biopsies, and/or microdissected tissue samples. Therefore, linear amplification protocols to increase the amount of RNA have been developed. The correlation between the results of microarray experiments derived from non-amplified RNA and amplified samples needs to be evaluated in detail. Methods: Total RNA was amplified and replicate hybridisation experiments were performed with linearly amplified (aRNA) and non-amplified mRNA from tonsillar B cells and the SUDHL-6 cell line using cDNA microarrays containing approximately 4500 genes. The results of microarray differential expression using either source of RNA (mRNA or aRNA) were also compared with those found using real time quantitative reverse transcription polymerase chain reaction (QRT-PCR). Results: Microarray experiments using aRNA generated reproducible data displaying only small differences to data obtained from non-amplified mRNA. The quality of the starting total RNA template and the concentration of the promoter primer used to synthesise cDNA were crucial components of the linear amplification reaction. Approximately 80% of selected upregulated and downregulated genes identified by microarray analysis using linearly amplified RNA were confirmed by QRT-PCR using non-amplified mRNA as the starting template. Conclusions: Linear RNA amplification methods can be used to generate high fidelity microarray expression data of comparable quality to data generated by microarray methods that use non-amplified mRNA samples.

The p38 mitogen-activated protein kinase (MAPK) is a key mediator of stress, extracellular-, growth factor-, and cytokine-induced signaling, and has been implicated in the development of cancer. Our previous work showed evidence for p38 MAPK activation in a subset of transformed follicular lymphomas (Elenitoba-Johnson et al. An important aim of functional genomic research is to globally identify and quantify specific proteomic and/or transcriptomic changes that are associated with physiologic or diseased states, thus enabling the elucidation of genes/proteins and signaling pathways that are associated with the phenotypic expression of a particular cellular state. The currently available microarray technologies permit the quantification of tens of thousands of gene transcripts and have been largely successful in demonstrating the global transcriptional changes accompanying the transition between cellular quiescence and activation, or during transition from normal to pathologic states (1-7). Proteomics, on the other hand, allows identification and quantification of up to a few thousand proteins, limited by the currently available technologies and the complex nature of the proteome especially in higher eukaryotes and mammalian cell systems. While the most widely used proteomics approach is the two-dimensional (2-D) 1 gel-based method followed by MS (8 -10), the recent development of multidimensional liquid chromatographic methods combined with MS/MS (LC-LC-MS/MS) has permitted sensitive detection of low-abundance proteins, membrane proteins and proteins with extreme pI (11-15). The ability to perform global quantitative proteomics has been significantly enhanced by the advent of the ICAT-based technology that is efficient in simplifying the proteome, and in combination with threedimensional 3-D LC-MS/MS permits detection and quantifiFrom the ‡Associated Regional and University Pathologists (ARUP)

The anaplastic lymphoma kinase (ALK) on 2p23 is a tyrosine kinase that forms chimeric fusions with numerous translocation partners. We describe a mass spectrometry-based approach for the identification of ALK fusion partners. This approach accurately identified the nucleophosmin (NPM)-ALK fusion protein in an anaplastic large cell lymphoma (ALCL)-derived cell line carrying the t(2;5)(p23;q35), and the TPM3-ALK in a clinical biopsy of inflammatory myofibroblastic tumor (IMT) carrying the t(1;2)(q21;p23). This study shows the ability of mass spectrometry to identify oncogenic chimeric proteins resulting from chromosomal rearrangements. This strategy can be adapted for the identification of known and unknown translocation partners of chimeric ALK fusion proteins involved in oncogenesis.immunoprecipitation ͉ tandem mass spectrometry ͉ peptide mapping ͉ multiple enzyme digestion

Summary We have studied the expression of RHOF, a member of the Rho‐GTPase family, in an array of lymphoid cells and tissues. Previous microarray studies demonstrated RHOF upregulation in a subset of transformed follicular lymphomas. Real‐time quantitative polymerase chain reaction evaluated RHOF expression in lymphocyte subpopulations, and normal and malignant lymphoid tissue. Cells and tissues of B‐cell origin expressed higher RHOF levels than their T‐cell counterparts. Neoplastic cells and tissues of B‐cell origin expressed higher levels of RHOF than their benign cellular counterparts. Relatively elevated levels of RHOF were seen in lymphomas derived from germinal centre origin.