Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Methodology U937 Human Immune Cells Control (No treatment) (n=4) Estrogen (5 uM) (n=4) 4-nonylphenol (5 uM) (n=4) Cultured Cells, RNA Isolation, RT (to cDNA) Applied cDNA to microarray chips Scanned chips for detection of gene expression by chemiluminescence Data analyzed by Spotfire software RT-PCR (using ER-beta) Reference gene: B-actin 2% agarose gel electrophoresis Spotfire Graphical Representation of Significantly Altered Genes Comparison of Altered Genes (By at least 2-Fold from Control) B A E1 4-NP Estrogen All genes from the wholegenome nanochips (15,960/33,155) Total Genes altered by 4-NP 14,913 414 633 (43.4%) (2.6%) (4.0%) Total Genes altered by estrogen C D E2 4-NP Estrogen Genes from customized portfolio (69/90) Significantly altered genes by 4-NP 54 7 8 (78.3%) (10.1%) (11.6%) Significantly altered genes by estrogen A/B) Scatterplots that represent Total gene expression analyzed by DNA microarray. The gene ID is represented by the x-axis and the fold change in gene expression are represented by the y-axis. (C/D) Scatterplots represent Only the significantly altered genes exhibiting at least a 2-fold-change from control. The x-axis represents the gene ID and the y-axis represents fold change in gene expression. (E1) Venn Diagram shows the numerical comparison of the total genes altered by 4-NP and estrogen as depicted in Figure A/B. (E2) Venn Diagram shows numerical representations of the gene expression of only the 90 portfolio genes. Portfolio genes include a total of 90 genes involved in estrogen signaling and breast cancer initiation. Signal-to-Noise Values of Four Genes Involved in Estrogen Signaling F1 F2 Probe_ID ERα ERβ Gene Symbol Gene Name Control E2 5µM 4NP 5µM 147628 ERα estrogen receptor alpha 3.39 -0.56 5.23 202485 ERβ estrogen receptor beta 13.22 2.34 1.87 171023 ERRβ estrogen-related receptor beta 46.7 32.35 29.92 (F1) 4-NP and estrogen have the potential to down regulate the estrogen receptor beta (ERβ) and the estrogen receptor related beta (ESRR-β) and up regulate repressor estrogen receptor activity (REA). ERα does not exhibit a significant pattern. (F2) Chart depicting numerical values for up/down regulation of gene after treatment. ERβ gene was used towards RTPCR validation step. ERRβ RT-PCR Gene Expression Profile Statistical Significance β-actin ERβ RT-PCR Gene Profile of ERβ G2 (G1) ERβ gene expression. Lane 1=control, Lane 2 =E2 5uM, Lane 3=control and Lane 4 = 4-NP 5uM. (G2) β-actin reference gene expression. Lane 1=control, Lane 2 =E2 5uM, Lane 3=control and Lane 4 = 4-NP 5uM. 1 2 3 4 1 2 3 4 mRNA/B-actin mRNA (%) G1 G3 140 120 100 80 60 40 20 0 n=4 n=4 n=4 Control (No Treatment) E2 (5 uM) Treatments 4-NP (5 uM) (G3) Graphical representation of RTPCR Analysis of Intensity of ERβ gene expression normalized with the internal control gene β-actin. This step serves as a validation for the microarray data.