Journal: BMC Evolutionary Biology

Article Title: Comparative analyses of glycerotoxin expression unveil a novel structural organization of the bloodworm venom system

doi: 10.1186/s12862-017-0904-4

Figure Lengend Snippet: Quantitative real-time PCR (qPCR) expression levels (shown as Fold Change, RQ) between biological groups (putative venom glands (pvg), pharyngeal lobes, and posterior body wall) of five analyzed GLTx transcripts (GLTx paralog 1–3, and adjacent gene regions GLTx-3’ and GLTx-5’; for details see Material and methods section “Quantitative real-time PCR”) in G. tridactyla ( n = 10; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05). a Relative GLTx expression (logarithmic scale) in putative venom glands (grey) and pharyngeal lobes (orange) in comparison to the GLTx expression signal exhibited by the body tissue (RQ = 1). Relative GLTx expression in the pharyngeal lobes and putative venom glands is significantly different from the expression signal in the body tissue. b Relative GLTx expression (linear scale) within the pharyngeal lobes in comparison to the putative venom glands (RQ = 1). Relative GLTx expression is significantly different between both putative venom glands and pharyngeal lobes

Article Snippet: Quantitative real-time PCR (qPCR) experiments were performed on Glycera tridactyla Schmarda, 1861 (Annelida, Glyceridae) specimens obtained from the Roscoff marine biological station in February 2015.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Comparison

Journal: Blood

Article Title: Mammalian target of rapamycin regulates neutrophil extracellular trap formation via induction of hypoxia-inducible factor 1 alpha

doi: 10.1182/blood-2012-01-405993

Figure Lengend Snippet: HIF-1α protein accumulation is posttranscriptionally regulated by mTOR in LPS-stimulated human PMNs. (A) We assessed HIF-1α (top) and IL-8 (bottom) transcript expression with the use of RNA-seq in control and stimulated human PMNs isolated from a single healthy adult donor. (B) Using RNA-seq, we determined the sequence of the 5′-UTR for the transcript coding for HIF-1α and predicted both its secondary structure (image) and the energy required to resolve its secondary structure (−182.4 kcal/mole).22 (C) qPCR was used to assess HIF-1α mRNA transcript expression in PMNs isolated from 3 different healthy adults at baseline and after LPS-stimulation over 1 hour. The results are presented as fold change over baseline ± SEM with baseline HIF-1α mRNA expression arbitrarily set at 1 (dashed line). One-way ANOVA analysis found no statistically significant differences. (D) We determined HIF-1α protein accumulation using In-Cell Western analysis for PMNs isolated from healthy adults at baseline and after LPS-stimulation for 1 hour with or without 1-hour pretreatment with rapamycin (200nM) or cycloheximide (500nM). The results are presented as fold change over baseline with baseline HIF-1α expression arbitrarily set at 1. The 1-way ANOVA with Tukey Multiple Comparisons posttesting was used. These results are representative of 5 separate experiments performed with human PMNs isolated from 5 different healthy adult donors. Statistical significance with *P < .05 and **P < .001. (E) We again assessed HIF-1α protein accumulation using In-Cell Western techniques in PMNs isolated from healthy adults at baseline and after LPS-stimulation for 1 hour with or without 1-hour pretreatment with rapamycin (200nM), torin 1 (250nM), or cycloheximide (500nM). The results are presented as absolute fluorescence normalized to cell number and are representative of 2 separate experiments performed with human PMNs isolated from 2 different healthy adult donors. (F) Human PMNs were stimulated with PAF (10nM) for 1 hour after a 1-hour pretreatment with or without rapamycin (200nM) or FK-506 (200nM). HIF-1α protein expression was quantified with semiquantitative immunocytochemistry, and the columns represent the mean fold change over baseline ± SEM. Control HIF-1α protein expression was arbitrarily set at 1 (dashed line). We used the single tailed Student t test for statistical analysis. *Significant difference (P < .05) between samples from PAF-stimulated PMNs and rapamycin-pretreated, PAF-stimulated PMNs. These experiments were performed with PMNs isolated from 3 different healthy adult donors.

Article Snippet: HIF-1 α transcript characterization using RNA-seq and qPCR in PMNs and surrogate PMNs The expression and sequence of mRNA coding for HIF-1 α in adult human PMNs were determined with paired-end next-generation RNA sequencing, as previously described with the use of the Illumina GAIIx sequencer with the assistance of the University of Utah Bioinformatics core, 21 and quantitative PCR (qPCR) with the following primer sets: HIF-1 α sense (5′-agagccgcttcatttcttaga-3′) and antisense (5′-tatccaaatcaccagcatcca-3′) and GAPDH sense (5′-gaacatcatccctgcctctactg-3′) and antisense (5′-agcttgacaaagtggtcgttgag-3′).

Techniques: Expressing, RNA Sequencing, Control, Isolation, Sequencing, In-Cell ELISA, Fluorescence, Immunocytochemistry

Journal: Viruses

Article Title: High-Throughput Screening of Antiviral Compounds Using a Recombinant Hepatitis B Virus and Identification of a Possible Infection Inhibitor, Skimmianine

doi: 10.3390/v16081346

Figure Lengend Snippet: Effect of skimmianine on HBV replication in HepG2.2.15 cells. HepG2.2.15 cells were seeded in 96-well plates, and 24 h later, skimmianine was added at 10 nM, 100 nM, 1 μM and 10 μM; entecavir at 200 nM; and DMSO at 2%. The medium was then replaced with fresh medium containing fresh compound and collected for the quantitation of the HBV-DNA level. On days 7, 10 and 13, the extracellular HBV-DNA level was quantitated by quantitative PCR and cell viability was analyzed on day 13 by a WST-8 assay. Each condition was tested using three wells of a 96-well plate. ( a ) HBV-DNA level in each condition. ( b ) HBV-DNA levels on day 10. Error bars show the standard deviation from the three wells. Each symbol shows the individual measurements. The differences in the means between 2% DMSO and each condition were analyzed with one-way analysis of variance with Bonferroni’s multiple comparisons test. **** p < 0.0001. ( c ) Cell viability relative to 2% DMSO. Each symbol shows the measurements. The differences in the means between 2% DMSO and each condition were analyzed with one-way analysis of variance with Bonferroni’s multiple comparisons test. ETV, entecavir; Skim, skimmianine; ns, not significant.

Article Snippet: Extracellular HBV-DNA levels were determined using an HBV Quantitative PCR (qPCR) Kit (Kubix, Hakusan, Japan) according to the manufacturer’s instructions.

Techniques: Quantitation Assay, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Viruses

Article Title: High-Throughput Screening of Antiviral Compounds Using a Recombinant Hepatitis B Virus and Identification of a Possible Infection Inhibitor, Skimmianine

doi: 10.3390/v16081346

Figure Lengend Snippet: Inhibitory effect of skimmianine on viral entry for non-recombinant HBV. Non-recombinant HBV from genotype C, designated WT-HBV, to which the HiBiT coding sequence was not inserted, was used to infect PXB cells. Then, skimmianine at concentrations ranging from 10 pM to 100 nM was added in both the entry and replication protocols, as shown in a. The medium was replaced with fresh medium containing fresh skimmianine every 3 days, and then the amounts of HBsAg ( a ) and HBV-DNA ( b ) in the medium were quantitated by ELISA and quantitative PCR, respectively, on day 21 after infection. Each symbol shows the individual measurements. Differences in the means between DMSO treatment and each treatment were analyzed via a one-way analysis of variance test. * p < 0.05, ** p < 0.01, and **** p < 0.0001. ns, not significant.

Article Snippet: Extracellular HBV-DNA levels were determined using an HBV Quantitative PCR (qPCR) Kit (Kubix, Hakusan, Japan) according to the manufacturer’s instructions.

Techniques: Recombinant, Sequencing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Infection

Journal: Viruses

Article Title: High-Throughput Screening of Antiviral Compounds Using a Recombinant Hepatitis B Virus and Identification of a Possible Infection Inhibitor, Skimmianine

doi: 10.3390/v16081346

Figure Lengend Snippet: Effect of skimmianine on attachment and internalization. HepG2-NTCP-YFP cells or PXB cells were pre-chilled on ice for 15 min and then incubated with 20,000 gEq/cell HBV derived from HepAD38 cells at 4 °C for 1.5 h either with indicated concentrations of skimmianine or without skimmianine (DMSO 2%). Heparin was used as a negative control at a final concentration of 100 IU/mL. After incubation, for attachment assay, cells were vigorously washed 3 times to remove free HBV. For internalization assay, cells were cultured at 37 °C for 6 h, and then they were trypsinized and washed 3 times to remove cell-attached HBV. Each DNA sample was extracted and quantified by quantitative PCR and normalized to 100 ng total DNA. ( a ) The results from HepG2-NTCP-YFP. ( b ) Those from PXB cells. Each symbol shows the individual measurements. Differences in the means between DMSO treatment and each treatment were analyzed via a one-way analysis of variance test. **** p < 0.0001. ns, not significant.

Article Snippet: Extracellular HBV-DNA levels were determined using an HBV Quantitative PCR (qPCR) Kit (Kubix, Hakusan, Japan) according to the manufacturer’s instructions.

Techniques: Incubation, Derivative Assay, Negative Control, Concentration Assay, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Journal of Gastrointestinal Oncology

Article Title: Expression and clinical significance of miR-17-5p in tumor tissues of patients with colorectal cancer

doi: 10.21037/jgo-22-1185

Figure Lengend Snippet: The effects of miR-17-5p on the invasive ability of HCT-116 cells. (A) Microscopic observation and photography after crystalline violet staining (100× magnification); (B) the OD values of each group at 570 nm wavelength after dissolution of crystalline violet by sodium acetate; (C-G) the relative mRNA and protein expression of miR-17-5p in HCT-116 cells was detected with RT-qPCR and western blot, respectively. *, P<0.05; **, P<0.01; ***, P<0.001. NC, negative control; in-NC, inhibitor negative control; RT-qPCR, real-time fluorescence quantitative polymerase chain reaction; OD, optical density.

Article Snippet: The miR-17-5p overexpression mimics, inhibitor, and corresponding controls, hairpin miRNAs quantification kits, and U6 calibration real-time fluorescence quantitative PCR (RT-qPCR) kits were purchased from Genepharma Co. (Shanghai, China).

Techniques: Staining, Dissolution, Expressing, Quantitative RT-PCR, Western Blot, Negative Control, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Journal of Gastrointestinal Oncology

Article Title: Expression and clinical significance of miR-17-5p in tumor tissues of patients with colorectal cancer

doi: 10.21037/jgo-22-1185

Figure Lengend Snippet: The effects of miR-17-5p on the apoptosis of HCT-116 cells. (A,B) Flow cytometric detection of the effects of different transfection groups on apoptosis of HCT-116 cells; (C-I) the relative mRNA and protein expression of miR-17-5p in HCT-116 cells was detected with RT-qPCR and Western blot, respectively. *, P<0.05; **, P<0.01; ***, P<0.001. NC, negative control; in-NC, inhibitor negative control; RT-qPCR, real-time fluorescence quantitative polymerase chain reaction.

Article Snippet: The miR-17-5p overexpression mimics, inhibitor, and corresponding controls, hairpin miRNAs quantification kits, and U6 calibration real-time fluorescence quantitative PCR (RT-qPCR) kits were purchased from Genepharma Co. (Shanghai, China).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Negative Control, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Frontiers in Physiology

Article Title: Ligand-binding properties of XaffOBP9, a Minus-C odorant-binding protein from Xyleborus affinis (Coleoptera: Curculionidae: Scolytinae)

doi: 10.3389/fphys.2023.1326099

Figure Lengend Snippet: The expression profiles of XaffOBPs using RNA-seq and RT-qPCR. Different lowercase letters indicate the significant differences in the expression level of XaffOBP s genes measured by RT-qPCR.

Article Snippet: The Real-time quantitative PCR (RT-qPCR) primers for the XaffOBPs genes were designed using Primer Premier v5.0 (Premier Biosoft, CA, United States) and are provided in . β-actin was employed as an internal reference.

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR