uantitative real-time polymerase chain reaction (PCR) is regarded as the gold standard for molecular profiling and target identification, but not in the context of high-throughput screening owing to limitations on workflow, cost of reagents, and miniaturization opportunities. Recent advances have moved reverse transcription quantitative PCR (RT-qPCR) forward, such as improvements in liquid handling, the launch of higher throughput platforms, and the release of one-step products. These one-step reagents enable the user to go straight from a cellular assay format to qPCR without the need for cumbersome and potentially expensive multistep RNA purification protocols. Our aim was to investigate the use of a one-step accelerated workflow to measure the levels of epidermal growth factor receptor (EGFR ) and nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2 ) gene expression using lysates generated by the RealTime ready Cell Lysis kit in downstream quantitative RT-qPCR. We present, for the first time, data from a vendor-independent one-step 1536 workflow that compares reporter gene and RT-qPCR screening approaches for oncology drug discovery. We also demonstrate a miniaturized and high-throughput workflow that could enable future application of this sensitive assay technology, with particular impact against phenotypic assays and those using rare cell types.

Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis.

Development of human placenta involves the invasion of trophoblast cells from anchoring villi into the maternal decidua. Placental transcription factor GCM1 regulates trophoblast cell invasion via transcriptional activation of HtrA4 gene, which encodes a serine protease enzyme. The GATA3 transcription factor regulates trophoblast cell differentiation and is highly expressed in invasive murine trophoblast giant cells. The regulation of trophoblastic invasion by GCM1 may involve novel cellular factors. Here we show that GATA3 interacts with GCM1 and inhibits its activity to suppress trophoblastic invasion. Immunohistochemistry demonstrates that GATA3 and GCM1 are coexpressed in villous cytotrophoblast cells, syncytiotrophoblast layer, and extravillous trophoblast cells of human placenta. Interestingly, GATA3 interacts with GCM1, but not the GCM2 homologue, through the DNA-binding domain and first transcriptional activation domain in GCM1 and the transcriptional activation domains and zinc finger 1 domain in GATA3. While GATA3 did not affect DNA-binding activity of GCM1, it suppressed transcriptional activity of GCM1 and therefore HtrA4 promoter activity. Correspondingly, GATA3 knockdown elevated HtrA4 expression in BeWo and JEG-3 trophoblast cell lines and enhanced the invasion activities of both lines. This study uncovered a new GATA3 function in placenta as a negative regulator of GCM1 activity and trophoblastic invasion.

Sulforaphane (SFN) is present in plants belonging to Cruciferae family and was first isolated from broccoli sprouts. Chemotherapeutic and anticarcinogenic properties of sulforaphane were demonstrated, however, the underlying mechanisms are not fully understood. In this study we evaluated the expression of cyclin D1 and p21 protein in SFN-treated A549 cells and correlated these results with the extent of cell death and/orcell cycle alterations, as well as determined a potential contribution of cyclin D1 to cell death. A549 cells were treated with increasing concentrations of SFN (30, 60 and 90 µM) for 24 h. Morphological and ultrastructural changes were observed using light, transmission electron microscope and videomicroscopy. Image-based cytometry was applied to evaluate the effect of SFN on apoptosis and the cell cycle. Cyclin D1and p21 expression was determined by flow cytometry, RT-qPCR and immunofluorescence. siRNA was used to evaluate the role of cyclin D1 in the process of suforaphane-induced cell death. We found that the percentage of cyclin D1-positive cells decreased after the treatment with SFN, but at the same time mean fluorescence intensity reflecting cyclin D1 content was increased at 30 µM SFN and decreased at 60 and 90 µM SFN. Percentage of p21-positive cells increased following the treatment, with the highest increase at 60 µM SFN, at which concentration mean fluorescence intensity of this protein was also significantly increased. The 30-µM dose of SFN induced an increased G2/M phase population along with a decreased polyploid fraction of cells, which implies a functional G2/M arrest. The major mode of cell death induced by SFN was necrosis and, to a lower degree apoptosis. Transfection with cyclin D1-siRNA resulted in significantly compromised fraction of apoptotic and necrotic cells, which suggests that cyclin D1 is an important determinant of the therapeutic efficiency of SFN in the A549 cells.