Genotyping assays often require substantial amounts of DNA. To overcome the problem of limiting amounts of available DNA, Whole Genome Amplification (WGA) methods have been developed. The multiple ...displacement amplification (MDA) method using Φ29 polymerase has become the preferred choice due to its high processivity and low error rate. However, the uniformity and fidelity of the amplification process across the genome has not been extensively characterized.
To assess amplification uniformity, we used array-based comparative genomic hybridization (aCGH) to evaluate DNA copy number variations (CNVs) in DNAs amplified by two MDA kits: GenomiPhi and REPLI-g. The Agilent Human CGH array containing nearly one million probes was used in this study together with DNAs from a normal subject and 2 cystic fibrosis (CF) patients. Each DNA sample was amplified 4 independent times and compared to its native unamplified DNA. Komogorov distances and Phi correlations showed a high consistency within each sample group. Less than 2% of the probes showed more than 2-fold CNV introduced by the amplification process. The two amplification kits, REPLI-g and GenomiPhi, generate very similar amplified DNA samples despite the differences between the unamplified and amplified DNA samples. The results from aCGH analysis indicated that there were no obvious CNVs in the CFTR gene region due to WGA when compared to unamplified DNA. This was confirmed by quantitative real-time PCR copy number assays at 10 locations within the CFTR gene. DNA sequencing analysis of a 2-kb region within the CFTR gene showed no mutations introduced by WGA.
The relatively high uniformity and consistency of the WGA process, coupled with the low replication error rate, suggests that WGA DNA may be suitable for accurate genotyping. Regions of the genome that were consistently under-amplified were found to contain higher than average GC content. Because of the consistent differences between the WGA DNA and the native unamplified DNA, characterization of the genomic region of interest, as described here, will be necessary to ensure the reliability of genotyping results from WGA DNA.
Reproducibility is a fundamental requirement in scientific experiments. Some recent publications have claimed that microarrays are unreliable because lists of differentially expressed genes (DEGs) ...are not reproducible in similar experiments. Meanwhile, new statistical methods for identifying DEGs continue to appear in the scientific literature. The resultant variety of existing and emerging methods exacerbates confusion and continuing debate in the microarray community on the appropriate choice of methods for identifying reliable DEG lists.
Using the data sets generated by the MicroArray Quality Control (MAQC) project, we investigated the impact on the reproducibility of DEG lists of a few widely used gene selection procedures. We present comprehensive results from inter-site comparisons using the same microarray platform, cross-platform comparisons using multiple microarray platforms, and comparisons between microarray results and those from TaqMan - the widely regarded "standard" gene expression platform. Our results demonstrate that (1) previously reported discordance between DEG lists could simply result from ranking and selecting DEGs solely by statistical significance (P) derived from widely used simple t-tests; (2) when fold change (FC) is used as the ranking criterion with a non-stringent P-value cutoff filtering, the DEG lists become much more reproducible, especially when fewer genes are selected as differentially expressed, as is the case in most microarray studies; and (3) the instability of short DEG lists solely based on P-value ranking is an expected mathematical consequence of the high variability of the t-values; the more stringent the P-value threshold, the less reproducible the DEG list is. These observations are also consistent with results from extensive simulation calculations.
We recommend the use of FC-ranking plus a non-stringent P cutoff as a straightforward and baseline practice in order to generate more reproducible DEG lists. Specifically, the P-value cutoff should not be stringent (too small) and FC should be as large as possible. Our results provide practical guidance to choose the appropriate FC and P-value cutoffs when selecting a given number of DEGs. The FC criterion enhances reproducibility, whereas the P criterion balances sensitivity and specificity.
Preclinical and clinical findings suggest sexual dimorphism in cardiotoxicity induced by a chemotherapeutic drug, doxorubicin (DOX). However, molecular alterations leading to sex‐related differential ...vulnerability of heart to DOX toxicity are not fully explored. In the present study, RNA sequencing in hearts of B6C3F1 mice indicated more differentially expressed genes in males than females (224 vs. 19; ≥1.5‐fold, False Discovery Rate FDR < 0.05) at 1 week after receiving 24 mg/kg total cumulative DOX dose that induced cardiac lesions only in males. Pathway analysis further revealed probable inactivation of cardiac apelin fibroblast signaling pathway (p = 0.00004) only in DOX‐treated male mice that showed ≥1.25‐fold downregulation in the transcript and protein levels of the apelin receptor, APJ. In hearts of DOX‐treated females, the transcript levels of apelin (1.24‐fold) and APJ (1.47‐fold) were significantly (p < 0.05) increased compared to saline‐treated controls. Sex‐related differential DOX effect was also observed on molecular targets downstream of the apelin‐APJ pathway in cardiac fibroblasts and cardiomyocytes. In cardiac fibroblasts, upregulation of Tgf‐β2, Ctgf, Sphk1, Serpine1, and Timp1 (fibrosis; FDR < 0.05) in DOX‐treated males and upregulation of only Tgf‐β2 and Timp1 (p < 0.05) in females suggested a greater DOX toxicity in hearts of males than females. Additionally, Ryr2 and Serca2 (calcium handling; FDR < 0.05) were downregulated in conjunction with 1.35‐fold upregulation of Casp12 (sarcoplasmic reticulum‐mediated apoptosis; FDR < 0.05) in DOX‐treated male mice. Drug effect on the transcript level of these genes was less severe in female hearts. Collectively, these data suggest a likely role of the apelin‐APJ axis in sex‐related differential DOX‐induced cardiotoxicity in our mouse model.
RNA sequencing in mouse hearts 1‐week after 24 mg/kg cumulative doxorubicin dose suggested a potential role of the apelin‐APJ pathway in sex‐related differential DOX‐induced cardiotoxicity. In males, the apelin receptor, APJ, was downregulated, whereas apelin and APJ were upregulated in females compared to saline‐treated counterparts. Downstream alterations were also seen, including significantly altered expression of Tgf‐β2, Sphk1, Serpine1, Ctgf, and Timp1 (fibrosis) in cardiac fibroblasts and Ryr2 and Serca2 (Ca2+ homeostasis) in cardiomyocytes with greater effects in males than females.
•HMB exposure differentially affected transcript levels in rat prostate and testis.•Gene expression changes in response to HMB were dose and tissue type dependent.•Drug metabolism pathways were ...altered by HMB exposure in the prostate and testis.•HMB exposure affected apoptosis and energy metabolism pathways in the testis.
2-hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet light-absorbing compound that is used in sunscreens, cosmetics and plastics. HMB has been reported to have weak estrogenic activity by in vivo and in vitro studies, making it a chemical with potential reproductive concern. To explore if prenatal and lactational HMB exposure alters gene expression profiles of the developing reproductive organs, we performed microarray analysis using the prostate and testis of postnatal day (PND) 30 male Sprague-Dawley rats offspring exposed to 0, 3000, or 30,000 ppm of HMB from gestational day 6 through PND 21. Gene expression profiles of the prostate and testis were differentially affected by HMB dose with significant alterations observed at the 30,000 ppm HMB group. Tissue-specific gene expression was also identified. These genes, whose expression was altered by HMB exposure, may be considered as candidate biomarker(s) for testicular or prostatic toxicity; however, further studies are necessary to explore this potential.
Pyrrolizidine alkaloids (PAs) are probably the most common plant constituents that poison livestock, wildlife, and humans worldwide. Riddelliine is isolated from plants grown in the western United ...States and is a prototype of genotoxic PAs. Riddelliine was used to investigate the genotoxic effects of PAs via analysis of gene expression in the target tissue of rats in this study. Previously we observed that the mutant frequency in the liver of rats gavaged with riddelliine was 3-fold higher than that in the control group. Molecular analysis of the mutants indicated that there was a statistically significant difference between the mutational spectra from riddelliine-treated and control rats.
Riddelliine-induced gene expression profiles in livers of Big Blue transgenic rats were determined. The female rats were gavaged with riddelliine at a dose of 1 mg/kg body weight 5 days a week for 12 weeks. Rat whole genome microarray was used to perform genome-wide gene expression studies. When a cutoff value of a two-fold change and a P-value less than 0.01 were used as gene selection criteria, 919 genes were identified as differentially expressed in riddelliine-treated rats compared to the control animals. By analysis with the Ingenuity Pathway Analysis Network, we found that these significantly changed genes were mainly involved in cancer, cell death, tissue development, cellular movement, tissue morphology, cell-to-cell signaling and interaction, and cellular growth and proliferation. We further analyzed the genes involved in metabolism, injury of endothelial cells, liver abnormalities, and cancer development in detail.
The alterations in gene expression were directly related to the pathological outcomes reported previously. These results provided further insight into the mechanisms involved in toxicity and carcinogenesis after exposure to riddelliine, and permitted us to investigate the interaction of gene products inside the signaling networks.
The development of nephrotoxicity limits the maximum achievable dosage and treatment intervals for cisplatin chemotherapy. Therefore, identifying mechanisms that regulate this toxicity could offer ...novel methods to optimize cisplatin delivery. MicroRNAs are capable of regulating many different genes, and can influence diverse cellular processes, including cell death and apoptosis. We previously observed miR-155 to be highly increased following ischemic or toxic injury to the kidneys and, therefore, sought to determine whether mice deficient in miR-155 would respond differently to kidney injury. We treated C57BL/6 and miR-155(-/-) mice with 20 mg/kg of cisplatin and found a significantly higher level of kidney injury in the miR-155(-/-) mice. Genome-wide expression profiling and bioinformatic analysis indicated the activation of a number of canonical signaling pathways relating to apoptosis and oxidative stress over the course of the injury, and identified potential upstream regulators of these effects. One predicted upstream regulator was c-Fos, which has two confirmed miR-155 binding sites in its 3' UTR and, therefore, can be directly regulated by miR-155. We established that the miR-155(-/-) mice had significantly higher levels of c-Fos mRNA and protein than the C57BL/6 mice at 72 h after cisplatin exposure. These data indicate a role for miR-155 in the cisplatin response and suggest that targeting of c-Fos could be investigated to reduce cisplatin-induced nephrotoxicity.
Sex is a risk factor for development of cardiotoxicity, induced by the anti-cancer drug, doxorubicin (DOX), in humans. To explore potential mechanisms underlying differential susceptibility to DOX ...between sexes, 8-week old male and female B6C3F1 mice were dosed with 3mg/kg body weight DOX or an equivalent volume of saline via tail vein once a week for 6, 7, 8, and 9 consecutive weeks, resulting in 18, 21, 24, and 27mg/kg cumulative DOX doses, respectively. At necropsy, one week after each consecutive final dose, the extent of myocardial injury was greater in male mice compared to females as indicated by higher plasma concentrations of cardiac troponin T at all cumulative DOX doses with statistically significant differences between sexes at the 21 and 24mg/kg cumulative doses. A greater susceptibility to DOX in male mice was further confirmed by the presence of cytoplasmic vacuolization in cardiomyocytes, with left atrium being more vulnerable to DOX cardiotoxicity. The number of TUNEL-positive cardiomyocytes was mostly higher in DOX-treated male mice compared to female counterparts, showing a statistically significant sex-related difference only in left atrium at 21mg/kg cumulative dose. DOX-treated male mice also had an increased number of γ-H2A.X-positive (measure of DNA double-strand breaks) cardiomyocytes compared to female counterparts with a significant sex effect in the ventricle at 27mg/kg cumulative dose and right atrium at 21 and 27mg/kg cumulative doses. This newly established mouse model provides a means to identify biomarkers and access potential mechanisms underlying sex-related differences in DOX-induced cardiotoxicity.
•Doxorubicin caused greater heart injury in male mice than females.•Doxorubicin caused vacuolization in cardiomyocytes only in male mice.•TUNEL-positive cardiomyocytes was higher in DOX-treated male mice.•γ-H2A.X-positive cardiomyocytes was greater in DOX-treated male mice.
Nonalcoholic fatty liver disease (NAFLD) is a major health problem and a leading cause of chronic liver disease in the United States and Western countries. In humans, genetic factors greatly ...influence individual susceptibility to NAFLD; nonetheless, the effect of inter-individual differences in the normal liver epigenome with regard to the susceptibility to NAFLD has not been determined.
In the present study, we investigated the association between the DNA methylation status in the livers of A/J and WSB/EiJ mice and the severity of NAFLD-associated liver injury. We demonstrate that A/J and WSB/EiJ mice, which are characterized by significant differences in the severity of liver injury induced by a choline- and folate-deficient (CFD) diet exhibit substantial differences in cytosine DNA methylation in their normal livers. Furthermore, feeding A/J and WSB/EiJ mice a CFD diet for 12 weeks resulted in different trends and changes in hepatic cytosine DNA methylation.
Our findings indicate a primary role of hepatic DNA methylation in the pathogenesis of NAFLD and suggest that individual variations in DNA methylation across the genome may be a factor determining and influencing the vulnerability to NAFLD.
Furan is a heterocyclic organic compound produced in the chemical manufacturing industry and also found in a broad range of food products, including infant formulas and baby foods. Previous reports ...have indicated that the adverse biological effects of furan, including its liver tumorigenicity, may be associated with epigenetic abnormalities. In the present study, we investigated the persistence of epigenetic alterations in rat liver. Male F344 rats were treated by gavage 5 days per week with 8 mg furan/kg body weight (bw)/day for 90 days. After the last treatment, rats were divided randomly into 4 groups; 1 group of rats was sacrificed 24 h after the last treatment, whereas other groups were maintained without further furan treatment for an additional 90, 180, or 360 days. Treatment with furan for 90 days resulted in alterations in histone lysine methylation and acetylation, induction of base-excision DNA repair genes, suggesting oxidative damage to DNA, and changes in the gene expression in the livers. A majority of these furan-induced molecular changes was transient and disappeared after the cessation of furan treatment. In contrast, histone H3 lysine 9 and H3 lysine 56 showed a sustained and time-depended decrease in acetylation, which was associated with formation of heterochromatin and altered gene expression. These results indicate that furan-induced adverse effects may be mechanistically related to sustained changes in histone lysine acetylation that compromise the ability of cells to maintain and control properly the expression of genetic information.
Microarray-based measurement of mRNA abundance assumes a linear relationship between the fluorescence intensity and the dye concentration. In reality, however, the calibration curve can be nonlinear.
...By scanning a microarray scanner calibration slide containing known concentrations of fluorescent dyes under 18 PMT gains, we were able to evaluate the differences in calibration characteristics of Cy5 and Cy3. First, the calibration curve for the same dye under the same PMT gain is nonlinear at both the high and low intensity ends. Second, the degree of nonlinearity of the calibration curve depends on the PMT gain. Third, the two PMTs (for Cy5 and Cy3) behave differently even under the same gain. Fourth, the background intensity for the Cy3 channel is higher than that for the Cy5 channel. The impact of such characteristics on the accuracy and reproducibility of measured mRNA abundance and the calculated ratios was demonstrated. Combined with simulation results, we provided explanations to the existence of ratio underestimation, intensity-dependence of ratio bias, and anti-correlation of ratios in dye-swap replicates. We further demonstrated that although Lowess normalization effectively eliminates the intensity-dependence of ratio bias, the systematic deviation from true ratios largely remained. A method of calculating ratios based on concentrations estimated from the calibration curves was proposed for correcting ratio bias.
It is preferable to scan microarray slides at fixed, optimal gain settings under which the linearity between concentration and intensity is maximized. Although normalization methods improve reproducibility of microarray measurements, they appear less effective in improving accuracy.
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