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RNA-seq Analysis
Published in Altuna Akalin, Computational Genomics with R, 2020
An MA plot is useful to observe if the data normalization worked well (Figure 8.6). The MA plot is a scatter plot where the x-axis denotes the average of normalized counts across samples and the y-axis denotes the log fold change in the given contrast. Most points are expected to be on the horizontal 0 line (most genes are not expected to be differentially expressed).
Analysis of DNA Microarrays in Clinical Trials
Published in Ding-Geng (Din) Chen, Karl E. Peace, Pinggao Zhang, Clinical Trial Data Analysis Using R and SAS, 2017
Ding-Geng (Din) Chen, Karl E. Peace, Pinggao Zhang
MA-plot. Another plot that has become widely used in microarray analysis is the MA-plot. It has been applied routinely as a part of a number of normalization procedures. MA-plots are typically used to compare two arrays or two groups of arrays. In a MA-plot, the vertical axis is the difference between the logarithms of the signals (i.e., the log ratio) and the horizontal axis is the average of the logarithms of the signals; “M” denotes minus and “A” denotes average.
Characterization of DNA hydroxymethylation profile in cervical cancer
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Jing Wang, Yi Su, Yongju Tian, Yan Ding, Xiuli Wang
Next, genomic DNA from four CSCC tissues of two I–IIa and two IIb–IV stage as well as two cervicitis were isolated and conducted 5mC and 5hmC immunoprecipitation approach combined with deep sequencing to map genome-wide 5mC and 5hmC profiles in cervical cancers. Approximately, 29.6 M reads of 5mC and 28.8 M reads of 5hmC sequencing data were collected, respectively (Table 1). To compare the individuals in cervical cancer and control groups, 5mC and 5hmC densities were normalized. Also, MA plot for a genome-wide comparison of 5mC/5hmC levels between IIb and IV stage CSCC and cervicitis groups displayed that most of the scatter plots are symmetrically distributed at the two sides of the 0 axis, which means that 5mC/5hmC sites were similar between the groups. Thus, no significant genome-wide differences in the 5mC/5hmC densities between the CSCC and cervicitis groups were observed (Figure 2(A,B)). After calling peaks by MACS software (p < .01), we mapped the (h)MeDIP-seq signals of 5mC and 5hmC peaks according to their genomic location and we did observe that 5mC was highly enriched at gene body and TES more than promoter regions (Figure 2(C)), while 5hmC was majorly enriched at the promoter and TSS regions (Figure 2(D)). The variation trend of the 5mC/5hmC levels had no significant difference among the three groups.
LILRB4 promotes tumor metastasis by regulating MDSCs and inhibiting miR-1 family miRNAs
Published in OncoImmunology, 2022
Mei-Tzu Su, Sakiko Kumata, Shota Endo, Yoshinori Okada, Toshiyuki Takai
Exosomes, with a size range of 30–150 nm in diameter, are an important part of the tumor microenvironment (TME).35 MicroRNAs (miRNAs) carried in exosomes are taken up by neighboring or distant cells to regulate cancer progression.36 To explore whether gp49B knockout affects the amount and composition of exosomal miRNAs, plasma exosomes from WT and gp49B−/− tumor-bearing mice were isolated for miRNA sequencing (miRNA-seq). An MA plot analysis revealed miRNAs that are differentially expressed between the WT miRNA-seq and gp49B−/− miRNA-seq (Figure 6a). By comparing the fold changes in gp49B−/− to that of WT samples, we observed that 4.46% miRNAs were upregulated while 3.80% miRNAs were downregulated in gp49B−/− plasma exosomes. Particularly, some of anti-tumor miRNAs showed higher fold changes in miRNA-seq data, including miR-1a-3p, miR-1b-5p, miR-133a-3p, and miR-206-3p (Figure 6a). We further validated that the expression levels of miR-1a-3p, miR-133a-3p, and miR-206-3p were increased in gp49B−/− plasma exosomes by qRT-PCR (Figure 6b). Moreover, the presence of gp49B−/− plasma exosomes from tumor-bearing mice indeed decreased the migration of LLC cells in vitro compared to the WT control (Figure 6, c and d). A comparative analysis revealed that the expression levels of miR-1a-3p and miR-133a-3p were significantly decreased in LUAD and LUSC tumor tissues compared to that in normal tissues (Supplemental Figure 6, A and B). The miR-206-3p expression was also downregulated in LUAD and LUSC samples, although with no statistical significance (Supplemental Figure 6C). These data suggested that LILRB4/gp49B contributes in the reduction of anti-tumor miRNAs in exosomes, which correlated with lung cancer diseases.
B cells require licensing by dendritic cells to serve as primary antigen-presenting cells for plasmid DNA
Published in OncoImmunology, 2023
Ichwaku Rastogi, Douglas G. McNeel
Next, we wished to understand the changes occurring in B cells at the gene expression level, following their interaction with DCs. DNA-loaded B cells were cultured for 3 days with DCs and CD8 T cells and then separated into individual populations by flow cytometry. B cells were then analyzed by RNAseq. Upon principal component analysis, the biological replicates demonstrated minimal variance, however large variation was observed between B cells cultured with DCs and those not cultured with DC (Figure 6a). This was indicative of vastly different gene expression signatures. This was confirmed by MA plot showing log fold change (M) of each gene plotted against its mean average intensity/expression (A) (Figure 6b). Similar analysis was also performed to analyze the gene expression variation in DCs before and after co-culture with DCs. PC plots and MA plots of the analysis showed that like B cells, DCs also demonstrated significant gene expression changes (6A-B). There were 6845 genes that were significantly (p < 0.05, adjusted for multiple comparisons) differentially regulated in B cells between the two groups. The top upregulated genes in B cells after co-culture with DCs were classified under the category of cytokine and chemokine related to immune system responses, more specifically related to inflammation type responses (Figures 6c and Supplemental Figure 4). We then performed gene set enrichment analysis (GSEA)20,21 to match this gene data set against prior defined B-cell related gene sets. Based on the enrichment scores and the relevance to APC function of B cells, we identified two prior defined gene sets most associated with DC-licensed B cells: B cells cultured with TLR7 agonist (imiquimod) versus TLR4 agonist (monophosphoryl lipid A) (Figure 6d,f), and B cells simulated through IgG (Figure 6E,g). Together, these gene sets suggested that the B cells licensed by DC had a gene expression profile consistent with an activated phenotype, similar to B cells activated by TLR and/or the B-cell receptor.