ABSTRACT
Death receptors (DRs) and the participants of DR-mediated signaling are characterized by a large number of mRNA isoforms generated by alternative splicing. Due to their high labor intensity and high cost, conventional methods (RT-PCR and RT-PCR in real time) are ineffective when the simultaneous detection of a plurality of mRNA isoforms is needed. In this regard, the use of DNA biochips is has prospective applications in analyzing the expression of many genes simultaneously. In this paper, we suggest an optimal strategy of probes selection aimed at detecting the maximum number of mRNA splice variants generated by major participants of DR-signaling. The objects of the study were 185 genes that form 1134 mRNA isoforms. As a result, a biochip design was developed that enables the detection of 499 mRNA isoforms (44% of total mRNA splice variants). The proposed strategy combines a high degree of modularity, the use of modern high-performance computers, and broad opportunities for setting up the selection criteria in accordance with the objectives of the study.
Subject(s)
Alternative Splicing , DNA Probes/metabolism , Molecular Probes/metabolism , RNA Precursors/metabolism , RNA, Messenger/metabolism , Receptors, Death Domain/genetics , Algorithms , Apoptosis/genetics , DNA Probes/chemistry , Exons , Humans , Introns , Molecular Probes/chemistry , Oligonucleotide Array Sequence Analysis , RNA Precursors/genetics , RNA, Messenger/genetics , Receptors, Death Domain/metabolism , Signal Transduction/geneticsABSTRACT
The CD38 gene codes a membrane protein which takes part in cell adhesion and catalyzes the formation of cyclic ADP-ribose. Using RT-PCR method we tested the presence of full-size and alternative forms of mRNA CD38 in samples of tumor tissue of patients with colorectal cancer and in tumor cell lines. It was shown that there are the cells in the tumor tissue which expressed CD38 gene. In tumor tissue of patients the alternative form of mRNA CD38 was detected less frequently than full-size form. Cells of lines Colo-205, T-84, HCT15 and HCT116 contained mRNA CD38, in cells of lines Caco-2 and SW-620 mRNA CD38 was absent. In cells of tumor tissue on the first stage of colorectal cancer CD38 gene was expressed in 100% of cases. On the second, third and fourth stages of the disease gene expression was observed less often. The frequency of mRNA CD38 detection not depend on tumor localization, tumor grade and presence of metastases. Using method of restriction analysis CpG methylation was detected in binding sites of transcription factor Sp1 and receptor of retinoic acid (RARE) in all tested samples of tumor tissue independently of the presence or absence of mRNA CD38. The obtained data suggest that in the tumor cells of patients with colorectal cancer the expression of the CD38 gene is heterogeneous.
