ABSTRACT: RNA-seq analysis of mRNAs and lncRNAs expression in chciken ALV-J-infected primary monocyte-derived macrophages (MDM) and uninfected control.
Project description:Purpose: The goals of this study are to investigate the differentially expressed miRNAs between ALV-J-infected primary monocyte-derived macrophages (MDM) and uninfected control by Illumina deep sequencing. Methods:Total RNA from two ALV-J-infected MDM (designated: J3h_1, J3h_2, J36h_1 and J36h_2) and two uninfected MDM samples (designated: NC3h_1, NC3h_2, NC36h_1 and NC36h_2) was isolated by TRIzol following the manufacturer’s instruction at 3 h post infection (hpi) and 36 hpi. RNA samples of two individuals within each group were pooled with equal amounts, and then were subjected to Illumina deep sequencing by Illumina Hiseq 2000. Results: compared to the uninfected MDM, we identified 13 significant up-regulated miRNAs and 2 significant down-regulated miRNAs in ALV-J infected MDM at 3 hpi, and 6 significant up-regulated miRNAs and 2 significant down-regulated miRNAs in ALV-J infected MDM at 36 hpi. Conclusions: Our results suggest that DE miRNAs involved in the immune response induced by ALV-J infection in MDM at 3 hpi. In addition, only 25 miRNAs-target DEGs were identified in MDM with ALV-J infection at 36 hpi, and these target DEGs can’t be significantly enriched in any GO terms and KEGG pathway..
Project description:Purpose: The goals of this study are to investigate the differentially expressed mRNAs and lncRNAs between ALV-J infected MDM and uninfected MDM in chickens by Illumina deep sequencing. Methods: Total RNA from two ALV-J-infected MDM (designated: J3h_1, J3h_2, J36h_1 and J36h_2) and two uninfected MDM samples (designated: NC3h_1, NC3h_2, NC36h_1 and NC36h_2) was isolated by TRIzol following the manufacturer’s instruction at 3 h post infection (hpi) and 36 hpi. RNA samples were subjected to Illumina deep sequencing by Illumina Hiseq 2000. Results:Compared to the uninfected control, a total of 1568 and 550 up-regulated genes were identified in chicken MDM at 3 hpi and 36 hpi respectively, and 1227 and 397 down-regulated genes were identified at 3 hpi and 36 hpi, respectively.128 and 30 DE lncRNAs were identified in MDM at 3 hpi and 36 hpi, respectively. Conclusions: Strong immune response induced by ALV-J infection in MDM at 3 hpi. Many genes, lncRNAs involved in immune response such as PRRs signaling pathway and Jak-STAT signaling pathway at 3 hpi. Specifically, 78 ISGs expression significantly increased in ALV-J-infected MDM at 3 hpi. We speculated that host innate immune response could inhibit ALV-J replication in chicken MDM. These results provide valuable insights into the game of host antiviral immune response and ALV-J infection.
Project description:We generated ribominus RNA sequencing data from three normal chicken spleen tissues and three ALV-J-infected chicken spleen tissues. We examined the expression of mRNAs in the spleens of 20-week-old chickens infected with ALV-J and uninfected chickens. 1723 mRNAs in the spleen showed differentially expressed between the uninfected and infected groups with a fold change ≥ 2 and P value < 0.05. Altered expression of several genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering, enrichment analysis of differentially expressed genes uncovered several tumor-associated or immune-related genes that may contribute to ALV-J-induced tumorigenesis in chicken.
Project description:Human alveolar macrophages (HAM) are primary bacterial niche and immune response cells during Mycobacterium tuberculosis (M.tb) infection, and human blood monocyte-derived macrophages (MDM) are a model for investigating M.tb-macrophage interactions. Here, we use a targeted RNA-Seq method to measure transcriptome-wide changes in RNA expression patterns of freshly obtained HAM (used within 6 h) and 6 day cultured MDM upon M.tb infection over time (2, 24 and 72 h), in both uninfected and infected cells from three donors each. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (AmpliSeq) uses primers targeting 18,574 mRNAs and 2,228 non-coding RNAs (ncRNAs) for a total of 20,802 transcripts. AmpliSeqTM yields highly precise and reproducible gene expression profiles (R2 >0.99). Taking advantage of AmpliSeq’s reproducibility, we establish well-defined quantitative RNA expression patterns of HAM versus MDM, including significant M.tb-inducible genes, in networks and pathways that differ in part between MDM and HAM. A similar number of expressed genes are detected at all time-points between uninfected MDM and HAM, in common pathways including inflammatory and immune functions, but canonical pathway differences also exist. In particular, at 2 h, multiple genes relevant to the immune response are preferentially expressed in either uninfected HAM or MDM, while the HAM RNA profiles approximate MDM profiles over time in culture, highlighting the unique RNA expression profile of freshly obtained HAM. MDM demonstrate a greater transcriptional response than HAM upon M.tb infection, with 2 to >10 times more genes up- or down-regulated. The results identify key genes involved in cellular responses to M.tb in two different human macrophage types. Follow-up bioinformatics analysis indicates that approximately 30% of response genes have expression quantitative trait loci (eQTLs in GTEx), common DNA variants that can influence host gene expression susceptibility or resistance to M.tb, illustrated with the TREM1 gene cluster and IL-10.
Project description:Relative expression levels of mRNAs in chicken cecal epithelia experimentally infected with Eimeria tenella were measured at 4.5 days post-infection. Two weeks old chickens were uninfected (negative control) or were orally inoculated with sporulated oocysts of Eimeria tenella. Cecal epithelia samples were collected from >12 birds in infected or uninfected group at 4.5 d following infections, in which samples from 4 birds were pooled together to form a total 3 biological replicates in each group. Parasite merozoites were also collected from four infected chickens at 5 d after infections. Uninfected control samples, merozoites and infection group samples were selected for RNA extraction and hybridization on Affymetrix microarrays. We used Affymetrix GeneChip chicken genome arrays to detail the chicken cecal epithelia gene expression in the control and E. tenella-infected birds.
Project description:In order to gain a better understanding of MAP infection in immature macrophages, high-throughput sequencing technology was used to perform an analysis of mRNAs profiles of bovine monocyte derived macrophages upon MAP infection. The total numbers of raw reads collected from the MAP-infected and control MDM cells were 93.6 and 102 million raw reads per library. After removing low-quality sequences, Adaptor sequences and sequences containing more than 5% N (N means that base information cannot be determined), 91.7 and 100 million clean reads were obtained respectively. The analyses of differential expression revealed 618 DE mRNAs in MDM challenged with MAP compared to the control MDM, Among the DE mRNAs, 322 were upregulated and 296 were downregulated.Furthermore, The GO and KEGG pathway enrichment of these mRNA targets were performed.
Project description:Relative expression levels of mRNAs in chicken cecal epithelia experimentally infected with Eimeria tenella were measured at 4.5 days post-infection. Two weeks old chickens were uninfected (negative control) or were orally inoculated with sporulated oocysts of Eimeria tenella. Cecal epithelia samples were collected from >12 birds in infected or uninfected group at 4.5 d following infections, in which samples from 4 birds were pooled together to form a total 3 biological replicates in each group. Parasite merozoites were also collected from four infected chickens at 5 d after infections. Uninfected control samples, merozoites and infection group samples were selected for RNA extraction and hybridization on Affymetrix microarrays. We used Affymetrix GeneChip chicken genome arrays to detail the chicken cecal epithelia gene expression in the control and E. tenella-infected birds. Infected, uninfected chicken cecal epithelia and merozoites were selected for RNA extraction and hybridization with Affymetrix microarrays. Our goal was to analyze global transcriptome changes in chicken cecal mucous membranes in response to E. tenella infection in vivo. We used infected (T1,T2,T3; three biological replicates) and uninfected (Neg1, Neg2, Neg3; three biological replicates) samples to identify genes that were differentially expressed. Meanwhile, RNA and probes were also prepared from parasite merozoites (Mzt) from infected samples (Mzt) and used as an additional control in microarray hybridization.
Project description:Avian leukosis virus (ALV) causes substantial economic losses from mortality and decreased performance in poultry industry. To characterize the response to ALV challenge, we developed a novel methodology that combines four datasets: mRNA expression and their associated regulatory factors of miRNA and lncRNA, and ALV gene expression. Specific Pathogen-Free (SPF) layer chickens were assigned to the ALV-infected or control group. Spleen samples (n=6) were collected at 40 days post injection (dpi), and sequenced. Comparing the infected and non-infected groups, 864 genes, 7 miRNAs and 17 lncRNAs were differentially expressed.
Project description:High quality CD14+ monocytes/macrophages (plMo/Mφ) were used for RNA-sequencing (RNA-seq) in comparison with human monocyte-derived macrophages (MDM) natural (MDM-0) or IL-4-polarized(MDM-IL4)