Protein microarray is a miniaturized multi-analyte, solid-phased immunoassay where thousands of immobilized individual protein spots on a microscopic slide bind are bound to specific antibodies (immunoglobulins) from serum samples, which are then detected by fluorescent labeling. The image processing and pattern recognition are then quantitatively analyzed using advanced algorithms. Here, we describe the use of an in-house-produced complex protein microarray containing extracts and pure proteins that has been probed with antibodies present in the horse sera and detection by fluorophore-conjugated antibody and data analysis. The flexibility of the number and types of proteins that can be printed on the microarray allows different set of specific IgE immunoassay analysis to be carried out.
Matched MeSH terms: Protein Array Analysis/methods
Most vertebrates possess at least two gonadotropin-releasing hormone (GnRH) neuron types. To understand the physiological significance of the multiple GnRH systems in the brain, we examined three GnRH neuron type-specific transcriptomes using single-cell microarray analyses in the medaka (Oryzias latipes). A microarray profile of the three GnRH neuron types revealed five genes that are uniquely expressed in specific GnRH neuron types. GnRH1 neurons expressed three genes that are homologous to functionally characterised genes, GnRH2 neurons uniquely expressed one unnamed gene, and GnRH3 neurons uniquely expressed one known gene. These genes may be involved in the modulation or maintenance of each GnRH neuron type.
Matched MeSH terms: Protein Array Analysis/methods*
Chemoresistance poses a major hurdle to cancer treatments. Andrographolide-derived SRJ09 and SRJ23 were reported to exhibit potent, selective inhibitory activities against colon and prostate cancer cells, respectively. In this study, previously developed resistant colon (HCT-116rst09) and prostate (PC-3rst23) cancer cell lines were used to elucidate the molecular mechanisms contributing to chemoresistance. Cytotoxic effects of SRJ09 and SRJ23 on both parental and resistant cells were investigated. Cell cycle distributions in HCT-116rst09 cells following SRJ09 treatment were analysed using flow cytometry. Whole-genome microarray analysis was performed on both parental and resistant cells to obtain differential gene expression profiles. Microarray data were subjected to protein-protein interaction network, functional enrichment, and pathway analyses. Reverse transcription-polymerase chain reaction (RT-PCR) was used to validate the changes in expression levels of selected genes. Besides morphological changes, HCT-116rst09 cells showed 7.0-fold resistance to SRJ09 while PC-3rst23 cells displayed a 5.5-fold resistance to SRJ23, as compared with their respective parental cells. G0/G1-phase cell cycle arrest was observed in HCT-116rst09 cells upon SRJ09 treatment. Collectively, 77 and 21 genes were found differentially modulated in HCT-116rst09 and PC-3rst23 cells, respectively. Subsequent bioinformatics analysis revealed several genes associated with FGFR4 and PI3K pathways, and cancer stemness, were chemoresistance mediators in HCT-116rst09 cells. RT-PCR confirmed the HMOX1 upregulation and ATG12 downregulation protected the PC-3rst23 cells from SRJ23 cytotoxicity. In conclusion, acquired chemoresistance to SRJ09 and SRJ23 in colon and prostate cancer cells, respectively, could be attributed to the alterations in the expression of genes such as those related to PI3K and autophagy pathways.
Matched MeSH terms: Protein Array Analysis/methods*
We previously found cytotoxic effects of tomato leaf extract (TLE) on the MCF-7 breast cancer cell line. The aim of this study was to ascertain the molecular mechanisms associated with the usage of TLE as an anticancer agent by microarray analysis using mRNA from MCF-7 breast cancer cells after treatment with TLE for 1 hr and 48 hrs. Approximately 991 genes out of the 30,000 genes in the human genome were significantly (p<0.05) changed after the treatment. Within this gene set, 88 were significantly changed between the TLE treated cells and the untreated MCF-7 cells (control cells) with a cut-off fold change >2.00. In order to focus on genes that were involved in cancer cell growth, only twenty-nine genes were selected, either down-regulated or up-regulated after treatment with TLE. Microarray assay results were confirmed by analyzing 10 of the most up and down regulated genes related to cancer cells progression using real-time PCR. Treatment with TLE induced significant up-regulation in the expression of the CRYAB, PIM1, BTG1, CYR61, HIF1-α and CEBP-β genes after 1 hr and 48 hrs, whereas the TXNIP and THBS1 genes were up-regulated after 1 hr of treatment but down-regulated after 48 hrs. In addition both the HMG1L1 and HIST2H3D genes were down-regulated after 1 hr and 48 hrs of treatment. These results demonstrate the potent activity of TLE as an anticancer agent.
Matched MeSH terms: Protein Array Analysis/methods