Journal: Genome Biology

Article Title: Global mRNA decay analysis at single nucleotide resolution reveals segmental and positional degradation patterns in a Gram-positive bacterium

doi: 10.1186/gb-2012-13-4-r30

Figure Lengend Snippet: Validation of the methods used for mRNA half-life estimation . (a-d) Validation was performed by calculation of the statistical correlation between half-lives for corresponding genes determined using different methods (a-c; B. cereus ATCC 10987), and statistical correlation between half-lives for orthologous genes from B. cereus strains ATCC 10987 and ATCC 14579, respectively (d), determined by RNA-Seq. Half-life values are plotted on log-scale. In each panel, the dashed line gives the regression line between two samples/tests/strains, and the number shown for each plot denotes the Pearson correlation for the gene orthologs. (a) Validation by RT-qPCR of half-lives determined by RNA-Seq (GA-II), demonstrating a good correlation of half-lives determined by the two methods. (b) Correlation between half-lives for the two biological replicate series B and D as determined by RNA-Seq (GA-II). (c) Correlation of mRNA half-lives determined by RNA-Seq, employing GA-II and 454 technology, respectively. (d) Correlation between half-lives of orthologous mRNAs in B. cereus strains ATCC 10987 and ATCC 14579, respectively.

Article Snippet: The other ATCC 14579 replicate (B series), however, showed a low correlation with RT-qPCR data (Pearson correlation 0.67, n = 6), as well as with the ATCC 14579 D series (correlation 0.60; Supplementary Figure S4 in Additional file ).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR

Journal: Journal of Applied Oral Science

Article Title: Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

doi: 10.1590/1678-775720150285

Figure Lengend Snippet: Forward and reverse primers used for CD25α, CCL, CCR, and transcription factor mRNA and 18S rRNA amplifications by qPCR

Article Snippet: The qPCR analysis for the CD25α mRNA expression in CD4+ T lymphocytes stimulated by autologous dendritic cells primed at a MOI=102 with the A. actinomycetemcomitans strains ATCC® 43717™ (serotype a), ATCC® 43718™ (serotype b), and ATCC® 43719™ (serotype c).

Techniques:

Journal: Journal of Applied Oral Science

Article Title: Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

doi: 10.1590/1678-775720150285

Figure Lengend Snippet: T-lymphocyte purification and activation. A: T-lymphocyte purification. Flow cytometry analysis of CD4, CD25α, CD45RA, and CD45RO expression demonstrating the purity of naïve CD4+ T lymphocytes isolated from healthy donors. B: T-lymphocyte activation. The qPCR analysis for the CD25α mRNA expression in CD4+ T lymphocytes stimulated by autologous dendritic cells primed at a MOI=102 with the A. actinomycetemcomitans strains ATCC® 43717™ (serotype a), ATCC® 43718™ (serotype b), and ATCC® 43719™ (serotype c). C: T-lymphocyte activation. Flow cytometry analysis of the CD25α expression demonstrating the levels of activation of CD4+ T lymphocytes after 5-day stimulation under the same conditions described in Figure 2B. The flow cytometry data from each experiment were expressed as percentage of positive cells over the total, and shown as mean±SD from 4 independent experiments. For relative expression, the CD25α mRNA expression in T lymphocytes cultured in the absence of dendritic cells was considered as 1, as a reference for fold-change in expression. Data are represented as fold-change for 8 independent experiments. Each experiment was performed in duplicate. Comparisons were done versus T lymphocytes exposed to non-induced dendritic cells (*p<0.05). Aa, Aggregatibacter actinomycetemcomitans ; CD, cluster of differentiation; DCs, dendritic cells

Article Snippet: The qPCR analysis for the CD25α mRNA expression in CD4+ T lymphocytes stimulated by autologous dendritic cells primed at a MOI=102 with the A. actinomycetemcomitans strains ATCC® 43717™ (serotype a), ATCC® 43718™ (serotype b), and ATCC® 43719™ (serotype c).

Techniques: Purification, Activation Assay, Flow Cytometry, Expressing, Isolation, Cell Culture

Journal: PLoS ONE

Article Title: Pdx1 and Ngn3 Overexpression Enhances Pancreatic Differentiation of Mouse ES Cell-Derived Endoderm Population

doi: 10.1371/journal.pone.0024058

Figure Lengend Snippet: (A, B) Tet-pdx1 ES cells were cultured according to Protocol #1. Pdx1 expressions were induced with Dox (closed circle, Dox + ) or without (open square, Dox − ) for 6 to 20 days. A) Gene expression was analyzed by RT-PCR. B) Ins1 mRNA levels were quantified by real time PCR and normalized to 18S mRNA levels. (C, D) EBs were differentiated for 6 days according to Protocol #1 and trypsinized to make single cell suspensions. The cells were electroporated with the pIRES2-EGFP vector and then reaggregated for 2–3 additional days with Dox. C) At day 8 (2 days with Dox), EGFP was evaluated by FACS. D) pIRES2 vectors, constructed to express GFP, Pax4, Nkx6.1 or Ngn3, were electroporated into day 6 EBs and allowed to reaggregate. At day 9, EBs were harvested and gene expression was analyzed by RT-PCR. (E, F) Tet-pdx1/ngn3 ES cells were cultured according to Protocol #1. Pdx1 and Ngn3 expression was induced with Dox (closed circle, Dox + ) or without (open square, Dox − ) at day 6 and harvested at the indicated time points. E) Ins1 mRNA levels were quantified by real time PCR and normalized to levels in βTC6 as described in the . F) Gene expression was analyzed by RT-PCR. Note, for the day 6 sample, the cells were exposed to Dox for only 1 hour before the samples were harvested.

Article Snippet: Quantitative PCR analysis revealed that the normalized (see ) level of Ins1 expression at day d20 represents 0.08% of the expression seen in the βTC6 insulinoma cell line (ATCC CRL-11506) ( ).

Techniques: Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Plasmid Preparation, Construct

Journal: PLoS ONE

Article Title: Pdx1 and Ngn3 Overexpression Enhances Pancreatic Differentiation of Mouse ES Cell-Derived Endoderm Population

doi: 10.1371/journal.pone.0024058

Figure Lengend Snippet: Tet-pdx1/ngn3 ES cells were cultured according to Protocol #2. Pdx1 and Ngn3 were induced with or without Dox starting at day 4, and cells were harvested at the indicated time points. (A–D) insulin and 18S mRNA levels were quantified by real time PCR and normalized to levels seen in βTC6 as described in the . A) Day 4 EBs were trypsinized and reaggregated with BMP4 (closed circles) or without (open squares). EBs were harvested at day 6 and 9. B) Day 4 EBs were trypsinized and reaggregated with BMP4. At day 6, EBs were replated on gelatin coated dishes and floating EBs were transferred to low-cluster dishes at day 7. Attached monolayer EBs (open bar) and floating EBs (closed bar) were harvested at day 9. (C) Floating EBs were cultured according to Protocol #2, with BMP4, and with Dox (circles) or without Dox (open squares). Ins1 (black circles) or Ins2 (white circles) mRNA levels were quantified by real time PCR and normalized to levels in βTC6 at the times indicated. (D) CXCR4/c-kit double negative (−/−) or double positive (+/+) cells were isolated by FACS from 4 days EBs, reaggregated for two days, and then replated on gelatin coated plates according to Protocol #2, with BMP4. Floating or attached EBs were harvested at day 9 from the following populations: pre-sorted cells (“Pre”); CXCR4/c-kit double negative (“−/−”); and CXCR4/c-kit double positive (“+/+”). These groups were cultured either as non-adherent EBs (“float”) or as attached monolayer cells (“mono”), as indicated.

Article Snippet: Quantitative PCR analysis revealed that the normalized (see ) level of Ins1 expression at day d20 represents 0.08% of the expression seen in the βTC6 insulinoma cell line (ATCC CRL-11506) ( ).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Isolation

Journal: PLoS ONE

Article Title: Pdx1 and Ngn3 Overexpression Enhances Pancreatic Differentiation of Mouse ES Cell-Derived Endoderm Population

doi: 10.1371/journal.pone.0024058

Figure Lengend Snippet: Tet-pdx1/ngn3 ES cells were cultured with Dox (Dox + ) or without (Dox − ) according to Protocol #3, including B27 supplement formulated with or without RA as indicated. (A) N2 supplement was added to the indicated groups from the beginning through day 14. B27(+RA), indicated by “+”, or B27(−RA), indicated by “−”, was included as a supplement from either day 0–4 or from day 4–14 as indicated. Ins1 mRNA levels at day 14 were quantified by real time PCR and normalized to βTC6 levels. (B) Tet-pdx1/ngn3 ES cells were cultured with Dox (Dox + ) or without (Dox − ) according to Protocol #3 for 18 days. EBs were trypsinized to make single cell suspensions and cytoplasmic insulin was detected by immunostaining and analyzed by FACS. (C) EBs were cultured according to Protocol #3 for 18 days with or without Dox, after which the media was replaced with fresh medium for 24 hours and supernatants were harvested. C-peptide, glucagon and somatostatin were measured by either RIA (C-peptide) or EIA (glucagon, somatostatin) as described in Materials & . (D) EBs cultured according to Protocol #3 for 19 days were resuspended in HKRB buffer and stimulated with KCl (30 mM), Forskolin (10 M) or IBMX (0.5 mM) for 1 hour. Supernatants were harvested and C-peptide was measured by RIA. For the Dox − samples in panels C & D, C-peptide and somatostatin values were not detectable above background. The data presented are mean of three independent experiments; the error bars represent the SEM. *p<0.05 as compared with Dox (−), **p<0.05 as compared with Dox(+).

Article Snippet: Quantitative PCR analysis revealed that the normalized (see ) level of Ins1 expression at day d20 represents 0.08% of the expression seen in the βTC6 insulinoma cell line (ATCC CRL-11506) ( ).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Immunostaining

Journal: PLoS ONE

Article Title: Prophage Induction and Differential RecA and UmuDAb Transcriptome Regulation in the DNA Damage Responses of Acinetobacter baumannii and Acinetobacter baylyi

doi: 10.1371/journal.pone.0093861

Figure Lengend Snippet: Delta Cq values from RT-qPCR experiments measuring expression of selected A. baumannii ATCC 17978 genes demonstrates the repressing activity of UmuDAb only for error prone polymerase components. The expression of each gene in both wild type and umuDAb null mutant is shown, with gene identity and A1S number listed on the x axis. Each gene was assayed in one RT-qPCR experiment (plate), with error bars indicating standard error of the mean from technical triplicates of biological triplicates.

Article Snippet: A. baylyi strains ADP1, ACIAD1385 (Δ recA ::KanR), and ACIAD2729 (Δ umuDAb ::KanR) as well as A. baumannii strains ATCC 17978, its isogenic recA insertion mutant , and a Δ umuDAb ::KanR null mutant were grown at 37°C in minimal media plus succinate for transcriptome and RT-qPCR analyses, and in Luria-Bertani broth for the production of phage particles.

Techniques: Quantitative RT-PCR, Expressing, Activity Assay, Mutagenesis