quantitative pcr rna Search Results


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  • 99
    Thermo Fisher rna ultrasense one step quantitative rt pcr system
    Dengue virus <t>RNA</t> replication in THP-1 cells. Cells were mock-infected or infected with DENV-3 at varying doses, incubated, and harvested after 1, 2 and 3 days post-infection. Total RNA from culture supernatant and cell lysates was extracted and reverse transcription quantitative real-time <t>PCR</t> was used to determine the expression of dengue virus mRNAs. Viral genome copy numbers in the supernatant ( A ) and cells ( B ) were estimated using dengue virus RNA as standards. Unpaired t -test was used to calculate significance (ns = no significance, * p ≤ 0.05 and *** p ≤ 0.001) vs. Day 1. §§ represents time-dependent variable.
    Rna Ultrasense One Step Quantitative Rt Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 523 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore qpcr total rna
    a Expression analysis of NFкB (RelA) and FAT1 in GBM samples . mRNA expression of NFкB (RelA) and FAT1 genes was analyzed in GBM tumors ( n = 16) with respect to the levels found in human normal brain total <t>RNA</t> (Clontech, USA) using <t>qPCR.</t> Increased expression of FAT1 was found in 9 out of 16 GBM samples as compared to the normal brain. High-NFkB expressors are represented as blue dots and low-NFkB expressors as black dots. FAT1 has been depicted with grey dots. b Correlation analysis of NFкB (RelA) and FAT1 expression in GBM samples . mRNA fold expression values of NFкB (RelA) and FAT1 found in the sixteen GBM tumors were correlated using Spearman’s analysis by SPSS 11.5. A significant positive correlation was observed between NFкB (RelA) and FAT1 ( r = 0.7, p = 0.002) in the studied samples. GBM samples have been displayed as high NFкB (RelA) expressors (blue dots) and low NFкB (RelA) expressors (black dots) on the basis of a cut-off value of ≥1.5 fold expression. c Correlation of NFкB (RelA) and FAT1 expression in Rembrandt GBM database . Expression values of NFкB (RelA) and FAT1 were obtained and correlated in 214 GBM cases belonging to Rembrandt database. A significant positive correlation was observed between NFкB (RelA) and FAT1 (Spearman’s ‘ r ’ = 0.276, p = 0.00004) in the analyzed cases. d Correlation of NFкB (RelA) and FAT1 expression in TCGA glioma database . Expression values of NFкB (RelA) and FAT1 were obtained and correlated in 153 glioma cases belonging to TCGA database. A significant positive correlation was observed between NFкB (RelA) and FAT1 (Spearman’s ‘ r ’ = 0.213, p = 0.008) in the analyzed cases
    Qpcr Total Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    tiangen biotech co quantitative real time pcr total rna
    The AGE-RAGE pathway triggers autophagic death of cardiomyocytes. (A) Viability of NRVMs was assessed by the CCK8 assay after treatment with various concentrations of AGE. (B) Cell viability measured by the CCK8 assay after AGE treatment (400 μg/ml) in NRVMs with RAGE overexpression or knockdown. (C) Expression of RAGE and Beclin 1 were detected by western blotting after AGE treatment of NRVMs with RAGE overexpression or knockdown. Representative figures are from different blots in Supplementary Figure S1 . (D,E) Quantitative analysis of RAGE and Beclin 1 protein levels. (F,G) NRVMs were infected with short hairpin <t>RNA</t> (shRNA) targeting ATG5, ATG7, or the scramble control. Expression of ATG5 and ATG7 was detected by <t>q-RT-PCR.</t> (H) Knockdown of ATG5 reduced the AGE-induced death of NRVMs. n = 3. Data are the mean ± SE., ∗ P
    Quantitative Real Time Pcr Total Rna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 92/100, based on 286 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    MACHEREY NAGEL quantitative real time pcr total rna
    ETV5 and SOX9 do not regulate N-cadherin ( A ) Transcriptional regulators deregulated in the presence of AR-V7 compared to AR-WT with known binding sites in CDH2 gene are listed. ( B ) IPA analysis from deregulated genes in the presence of AR-V7 highlighted a network in which ETV5 was a direct regulator of CDH2. Green: downregulated expression; Red: upregulated expression. ( C ) A lentiviral inducible system was used to verify ETV5 expression in the presence of AR variants observed in our <t>RNA-seq</t> data. AR-WT and AR variants expression were induced with 20 ng/mL doxycycline. LNCaP expressing AR-WT and AR variants were cultured in the presence of 10nM DHT and ETV5 mRNA expression level was analyzed by real-time <t>PCR</t> 4 days after induction. ( D ) To analyze the impact of ETV5 on N-cadherin expression, AR-WT and AR-V7 were induced in LNCaP with 20 ng/mL doxycycline and cells were transfected with 50 nM of siRNA against ETV5 (left panel). After 48 h, total mRNA was extracted and CDH2 mRNA expression level was assessed by qRT-PCR (right panel). For all qRT-PCR analyses, the results were normalized to β-ACTIN. Relative expression is represented as the mean of ΔΔCt ± SEM of three independent experiments. NS: not significant, *** P
    Quantitative Real Time Pcr Total Rna, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 93/100, based on 499 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad quantitative real time pcr total rna
    DNMT3B modulates exon 4–5–6 skipping by physically interacting with hnRNP-LL and CD45 pre-mRNA. ( A ) Native <t>RNA</t> immunoprecipitation assay (RIP-qPCR) shows DNMT3B interaction with CD45 pre-mRNA in control and ICF1 samples. Binding to HOXC4 mRNA is reported as negative control; ( B ) DNMT3B enrichment at DNA (exons 4, 5 and 6) of CD45 gene by ChIP assay; ( C and D ) Expression level of hnRNP-LL in ICF1 samples and controls by qPCR and western blot, respectively; ( E ) CpG methylation level [log2 (methylated/unmethylated)] at hnRNP-LL promoter in ICF1 samples; ( F ) DNMT3B and hnRNP-LL physically interact in ICF1p2 sample as shown in co-immunoprecipitation experiments; ( G ) Expression level (qPCR) of CD45RABC and the constitutive exon9 using isoform specific oligonucleotides upon siDNMT3B and control siRNAs transfection; ( H ) Semi-quantitative <t>PCR</t> amplification of CD45 splicing isoforms in overexpressing-hnRNP-LL ICF1p2 sample upon siDNMT3B and control siRNAs transfection. * P -adj
    Quantitative Real Time Pcr Total Rna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rna probes
    Co-infection of Gouleako goukovirus <t>(GOLV)</t> and RVFV. (A) C6/36 cells were mock-infected or infected with GOLV (8 MOI) and/or rMP-12 (0.5 MOI) at 28°C. Total <t>RNA</t> was extracted at 72 hpi (left four lanes). Culture supernatants were collected and transferred into either C6/36 cells (middle four lanes) or Vero cells (right four lanes). Total RNA was then extracted at 72 hpi. The presence of L-, M-, and S-segment RNA of GOLV (top panel) or RVFV (bottom panel) was analyzed by Northern blot. (B) Vero or PK15 cells were infected with GOLV (8 MOI) with or without either rMP-12 (0.5 MOI) or rMP12-ΔNSs16/198 (2.0 MOI) at 28°C. Total RNA was extracted at 18 hpi, and the accumulation of viral RNA was analyzed by Northern blot. Top panel: probes to detect negative-sense GOLV L-, M-, and S-segments, Bottom panel: probes to detect negative-sense RVFV L-, M-, and S-segments. GOLV RNA derived from C6/36 cells infected with GOLV (8 MOI) was used for the positive control for GOLV probes on the membrane.
    Rna Probes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 223 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    SABiosciences rt2 qpcr grade rna isolation kit
    <t>QPCR</t> custom array heat map of relative gene expression throughout the time course of acute (A) or chronic (B) cuprizone treatment and recovery <t>RNA</t> was isolated from the corpus callosum of mice at each time point shown along the top of the heat map. The time points are indicated as the number of weeks of cuprizone ingestion (0, 3, 6, 9 or 12) followed by the weeks on normal chow for recovery (+3 or +6). Results show fold-change values for each gene from triplicate PCR reactions which were each run on a separate plate. For each gene, relative expression levels are shown with the highest as dark red and the lowest as dark blue. Genes are hierarchically clustered based on correlation with expression of GFAP across each time course. Genes above the arrow on each heat map show a pattern similar to GFAP. The age-matched equivalent to the beginning of cuprizone treatment is shown as 0 week while the equivalent to the end of the time course of treatment and recovery is 20 week ( A ) for the acute and 26 week ( B ) for the chronic time course.
    Rt2 Qpcr Grade Rna Isolation Kit, supplied by SABiosciences, used in various techniques. Bioz Stars score: 89/100, based on 203 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher stabilized blood to ct nucleic acid preparation kit for qpcr
    Differential expression of glycogen synthesizing enzymes due to increased (p)ppGpp levels in Δ fumABC . A) STM WT harboring a dual color fluorescence reporter for wraB was cultured in LB o/n and subcultured in minimal medium with or without amino acid (= aa) supplementation (dashed or undashed line, respectively). After 3 h of growth cells were subjected to flow cytometry and sfGFP fluorescence intensity (BL1-H) recorded. Shown is one representative of three independent biological replicates. B) Representative data of WT, Δ fumABC , Δ fumABC Δ glgA and Δ relA Δ spoT strains harboring the wraB reporter grown o/n in LB broth. C) Medians of relative sfGFP fluorescence intensities of strains mentioned in (B). Data were normalized to WT (=1) and represent average values and standard deviation of three biological replicates. D) WT, Δ fumABC and Δ fumABC Δ glgA strains were cultured o/n in LB broth, <t>RNA</t> was extracted and used for cDNA synthesis and consecutive <t>qPCR</t> experiments. 16s rRNA expression levels were used for normalization. Depicted are the expression levels normalized to WT (=1) of glgA and glgC . Shown is one representative assay of three independent biological replicates, consisting each of three technical replicates. Statistical analyses were performed by Student’s t -test and significances are indicated as follows: *, p
    Stabilized Blood To Ct Nucleic Acid Preparation Kit For Qpcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Stratagene qpcr human reference total rna
    <t>qPCR</t> analysis of intact (T) and partly-degraded (Tf) <t>RNA</t> T using Transcriptor. Experimental setup and display are as in Fig. 1 . Arrows exemplify the trend of change in Cq values using the highlighted dilution (outlined circles).
    Qpcr Human Reference Total Rna, supplied by Stratagene, used in various techniques. Bioz Stars score: 89/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    PEQLAB quantitative real time pcr total rna
    Quantitative real-time <t>PCR</t> measuring mRNA expression of decidual marker genes in differentiating HDSC and THESC . Cultures were incubated with cAMP or E2P4 for 3, 6, 9 and 12 days. Cells without stimuli were kept in parallel representing non-stimulated controls (n.c.). <t>RNA</t> extraction and quantitative real-time PCR were performed as described in Methods. For relative quantification of mRNA expression n.c. of day 3 was arbitrarily set at 1 (calibrator). Bars indicate mean values ± SEM of seven (HDSC) and five (THESC) different experiments/PCR reactions performed in duplicates.* depicts p
    Quantitative Real Time Pcr Total Rna, supplied by PEQLAB, used in various techniques. Bioz Stars score: 93/100, based on 186 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rt qpcr total rna
    PACT Ser-246 and Ser-287 are necessary for PKR interaction and downstream inflammatory signaling. ( A ) PACT KO MEFs were reconstituted with FLAG-WT PACT or FLAG-S246A mutant PACT constructs, then treated with 600 mOsm sucrose for 3 hr. Proteins from total cell extracts or co-immunoprecipitated with the FLAG antibody were analyzed via western blot. ( B ) PACT KO MEFs were reconstituted with FLAG-WT PACT, FLAG-S246A, or FLAG-S287A mutant PACT constructs, then treated with 600 mOsm sucrose for 3 hr. Total lysates were analyzed via western blot. ( C ) PACT KO MEFs were reconstituted with FLAG-WT PACT or FLAG-S246A mutant PACT constructs, then treated with 600 mOsm sucrose for 3 hr. <t>RNA</t> was isolated, and Nos2 transcript levels were analyzed via <t>qPCR.</t> ( D ) PKR KO MEFs reconstituted with wild type PKR, PKR mutated at the RNA-binding residues (K64R/K154R), or kinase activity-deficient PKR (K296R) were treated with 500 or 600 mOsm sucrose for the indicated durations. RNA was isolated, and Nos2 transcript levels were analyzed via qPCR.
    Rt Qpcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5640 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Molecular Research Center inc quantitative rt pcr total rna
    Glutamine‐restriction improves the metabolic status in CD 8 + T cells. OVA ‐specific OT ‐1 CD 8 + T cells were cultured as shown in (Figure 1 A). A, The O 2 consumption rate ( OCR ) was measured in real time under basal conditions and in response to indicated mitochondrial inhibitors. The spare respiratory capacity ( SRC ) is shown (left panel). Data are representative of at least 2 independent experiments. B, The gene expression of mitochondrial transcription factors ( Tfam1 and Tfb2 m ) and <t>RNA</t> polymerase ( Polrmt ) in CD 8 + T cells cultured under glutamine‐restricted conditions. The expression of mRNA was examined by quantitative RT ‐ <t>PCR</t> and compared between control (Ctrl)‐cultured and dG ln‐cultured cells (mean ± SD , n = 4 per group). C, Representative flow‐cytometric profiles of mitochondrial reactive oxygen species (ROS) . Data are representative of 2 independent experiments. D, The extracellular acidification rate ( ECAR ) was measured under basal conditions and in response to 10 mmol/L glucose, 100 mmol/L glucose and 10 μmol/L oligomycin. Data are representative of 2 independent experiments. E, The gene expression of glycolysis‐related enzymes in CD 8 + T cells cultured under glutamine‐restricted conditions. The expression of mRNA was examined by quantitative RT ‐ PCR and compared between Ctrl‐ and dG ln‐cultured cells (mean ± SD , n = 4 per group). ** P
    Quantitative Rt Pcr Total Rna, supplied by Molecular Research Center inc, used in various techniques. Bioz Stars score: 92/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa qpcr human reference total rna
    Glutamine‐restriction improves the metabolic status in CD 8 + T cells. OVA ‐specific OT ‐1 CD 8 + T cells were cultured as shown in (Figure 1 A). A, The O 2 consumption rate ( OCR ) was measured in real time under basal conditions and in response to indicated mitochondrial inhibitors. The spare respiratory capacity ( SRC ) is shown (left panel). Data are representative of at least 2 independent experiments. B, The gene expression of mitochondrial transcription factors ( Tfam1 and Tfb2 m ) and <t>RNA</t> polymerase ( Polrmt ) in CD 8 + T cells cultured under glutamine‐restricted conditions. The expression of mRNA was examined by quantitative RT ‐ <t>PCR</t> and compared between control (Ctrl)‐cultured and dG ln‐cultured cells (mean ± SD , n = 4 per group). C, Representative flow‐cytometric profiles of mitochondrial reactive oxygen species (ROS) . Data are representative of 2 independent experiments. D, The extracellular acidification rate ( ECAR ) was measured under basal conditions and in response to 10 mmol/L glucose, 100 mmol/L glucose and 10 μmol/L oligomycin. Data are representative of 2 independent experiments. E, The gene expression of glycolysis‐related enzymes in CD 8 + T cells cultured under glutamine‐restricted conditions. The expression of mRNA was examined by quantitative RT ‐ PCR and compared between Ctrl‐ and dG ln‐cultured cells (mean ± SD , n = 4 per group). ** P
    Qpcr Human Reference Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Omega Bio-tek quantitative real time pcr total rna
    Linc-ROR shares miRNA binding sites with SOX9. a <t>qRT-PCR</t> analysis of the expression of linc-ROR and SOX9 in EC9706 cells transfected with 12 different miRNA mimics versus scramble control. b qRT-PCR analysis of linc-ROR and SOX9 expression after treatment with miR-145 inhibitor; a mixture of miR-15b, miR-33a, and miR-129 inhibitors (3 miR-inh mix); and inhibitor cocktail of miR-15b, miR-33a, miR-129, miR-145, and miR-206 (5 miR-inh mix). c qRT-PCR analysis of stemness-associated genes CD44, KLF4, NANOG, OCT4, and SOX2 expression following treatment with miR-145 mimics in EC9706 or 5 miR-inh mix in Eca109 cells. Transcription levels were normalized to GAPDH expression. d Prediction for five candidate miRNA-binding elements on linc-ROR transcript and SOX9 3′-UTR. e Sequence alignment of miR-15b, miR-33a, miR-129, miR-145, and miR-206 seed sequence in linc-ROR and SOX9 3′-UTR. f Amount of linc-ROR and SOX9 bound to Ago2 was determined by qRT-PCR in the presence of inhibitor cocktail of all candidate miRNAs or negative control. IgG was used as a negative control. g SOX9 protein level in EC9706 cells following treatment with linc-ROR siRNA or miR-145 mimics was measured by Western blot (top). Luciferase assay of 293 T cells co-transfected with pmiR-REPORT-SOX9 3′-UTR and indicated <t>RNA</t> oligos. siRNA targeting SOX9 3′-UTR was used as a positive control (bottom)
    Quantitative Real Time Pcr Total Rna, supplied by Omega Bio-tek, used in various techniques. Bioz Stars score: 90/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Dengue virus RNA replication in THP-1 cells. Cells were mock-infected or infected with DENV-3 at varying doses, incubated, and harvested after 1, 2 and 3 days post-infection. Total RNA from culture supernatant and cell lysates was extracted and reverse transcription quantitative real-time PCR was used to determine the expression of dengue virus mRNAs. Viral genome copy numbers in the supernatant ( A ) and cells ( B ) were estimated using dengue virus RNA as standards. Unpaired t -test was used to calculate significance (ns = no significance, * p ≤ 0.05 and *** p ≤ 0.001) vs. Day 1. §§ represents time-dependent variable.

    Journal: International Journal of Molecular Sciences

    Article Title: Secretion of Galectin-9 as a DAMP during Dengue Virus Infection in THP-1 Cells

    doi: 10.3390/ijms18081644

    Figure Lengend Snippet: Dengue virus RNA replication in THP-1 cells. Cells were mock-infected or infected with DENV-3 at varying doses, incubated, and harvested after 1, 2 and 3 days post-infection. Total RNA from culture supernatant and cell lysates was extracted and reverse transcription quantitative real-time PCR was used to determine the expression of dengue virus mRNAs. Viral genome copy numbers in the supernatant ( A ) and cells ( B ) were estimated using dengue virus RNA as standards. Unpaired t -test was used to calculate significance (ns = no significance, * p ≤ 0.05 and *** p ≤ 0.001) vs. Day 1. §§ represents time-dependent variable.

    Article Snippet: Determination of DENV Copy Number by Reverse Transcription Quantitative Real-Time PCR Viral copy numbers in culture supernatants and cell lysates were measured by qRT-PCR using the RNA UltraSense One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA, USA) and Thermal Cycler Dice Real Time System (Takara Bio, Otsu, Japan) according to the manufacturers’ protocols and as described previously [ ].

    Techniques: Infection, Incubation, Real-time Polymerase Chain Reaction, Expressing

    Relative expression of LGALS9 mRNA. THP-1 cells were mock-infected or infected with DENV-3 at varying doses. Cells were incubated and harvested after 1, 2 and 3 days post-infection. Total RNA from cell lysates was extracted and reverse transcription quantitative RT-PCR was used to determine the expression of LGALS9 mRNA. The mRNA levels of galectin-9 (Gal-9) were normalized to GAPDH mRNA.

    Journal: International Journal of Molecular Sciences

    Article Title: Secretion of Galectin-9 as a DAMP during Dengue Virus Infection in THP-1 Cells

    doi: 10.3390/ijms18081644

    Figure Lengend Snippet: Relative expression of LGALS9 mRNA. THP-1 cells were mock-infected or infected with DENV-3 at varying doses. Cells were incubated and harvested after 1, 2 and 3 days post-infection. Total RNA from cell lysates was extracted and reverse transcription quantitative RT-PCR was used to determine the expression of LGALS9 mRNA. The mRNA levels of galectin-9 (Gal-9) were normalized to GAPDH mRNA.

    Article Snippet: Determination of DENV Copy Number by Reverse Transcription Quantitative Real-Time PCR Viral copy numbers in culture supernatants and cell lysates were measured by qRT-PCR using the RNA UltraSense One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA, USA) and Thermal Cycler Dice Real Time System (Takara Bio, Otsu, Japan) according to the manufacturers’ protocols and as described previously [ ].

    Techniques: Expressing, Infection, Incubation, Quantitative RT-PCR

    a Expression analysis of NFкB (RelA) and FAT1 in GBM samples . mRNA expression of NFкB (RelA) and FAT1 genes was analyzed in GBM tumors ( n = 16) with respect to the levels found in human normal brain total RNA (Clontech, USA) using qPCR. Increased expression of FAT1 was found in 9 out of 16 GBM samples as compared to the normal brain. High-NFkB expressors are represented as blue dots and low-NFkB expressors as black dots. FAT1 has been depicted with grey dots. b Correlation analysis of NFкB (RelA) and FAT1 expression in GBM samples . mRNA fold expression values of NFкB (RelA) and FAT1 found in the sixteen GBM tumors were correlated using Spearman’s analysis by SPSS 11.5. A significant positive correlation was observed between NFкB (RelA) and FAT1 ( r = 0.7, p = 0.002) in the studied samples. GBM samples have been displayed as high NFкB (RelA) expressors (blue dots) and low NFкB (RelA) expressors (black dots) on the basis of a cut-off value of ≥1.5 fold expression. c Correlation of NFкB (RelA) and FAT1 expression in Rembrandt GBM database . Expression values of NFкB (RelA) and FAT1 were obtained and correlated in 214 GBM cases belonging to Rembrandt database. A significant positive correlation was observed between NFкB (RelA) and FAT1 (Spearman’s ‘ r ’ = 0.276, p = 0.00004) in the analyzed cases. d Correlation of NFкB (RelA) and FAT1 expression in TCGA glioma database . Expression values of NFкB (RelA) and FAT1 were obtained and correlated in 153 glioma cases belonging to TCGA database. A significant positive correlation was observed between NFкB (RelA) and FAT1 (Spearman’s ‘ r ’ = 0.213, p = 0.008) in the analyzed cases

    Journal: BMC Cancer

    Article Title: NFкB is a critical transcriptional regulator of atypical cadherin FAT1 in glioma

    doi: 10.1186/s12885-019-6435-1

    Figure Lengend Snippet: a Expression analysis of NFкB (RelA) and FAT1 in GBM samples . mRNA expression of NFкB (RelA) and FAT1 genes was analyzed in GBM tumors ( n = 16) with respect to the levels found in human normal brain total RNA (Clontech, USA) using qPCR. Increased expression of FAT1 was found in 9 out of 16 GBM samples as compared to the normal brain. High-NFkB expressors are represented as blue dots and low-NFkB expressors as black dots. FAT1 has been depicted with grey dots. b Correlation analysis of NFкB (RelA) and FAT1 expression in GBM samples . mRNA fold expression values of NFкB (RelA) and FAT1 found in the sixteen GBM tumors were correlated using Spearman’s analysis by SPSS 11.5. A significant positive correlation was observed between NFкB (RelA) and FAT1 ( r = 0.7, p = 0.002) in the studied samples. GBM samples have been displayed as high NFкB (RelA) expressors (blue dots) and low NFкB (RelA) expressors (black dots) on the basis of a cut-off value of ≥1.5 fold expression. c Correlation of NFкB (RelA) and FAT1 expression in Rembrandt GBM database . Expression values of NFкB (RelA) and FAT1 were obtained and correlated in 214 GBM cases belonging to Rembrandt database. A significant positive correlation was observed between NFкB (RelA) and FAT1 (Spearman’s ‘ r ’ = 0.276, p = 0.00004) in the analyzed cases. d Correlation of NFкB (RelA) and FAT1 expression in TCGA glioma database . Expression values of NFкB (RelA) and FAT1 were obtained and correlated in 153 glioma cases belonging to TCGA database. A significant positive correlation was observed between NFкB (RelA) and FAT1 (Spearman’s ‘ r ’ = 0.213, p = 0.008) in the analyzed cases

    Article Snippet: Expression analysis by qPCR Total RNA from treated and respective control cells was isolated using TRI reagent (Sigma, St Louis, MO).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Relative expression as well as H3K4me3 levels of conidiation‐related TF genes in Δ set1 and Δ kdm5 mutants. Indicated strains were grown on V8 agar in the presence of a 12 h light/12 h dark cycle for 3 days to induce conidiation. A . RNA was extracted and the relative expression of ABA1 , FLB3 and FLB4 was determined by RT‐qPCR. B . ChIP‐qPCR was performed for the same genes using the H3K4me3 antibody. Therefore, the enriched samples (precipitated by antibody) were normalized to the respective input samples (initially applied chromatin). The WT was arbitrarily set to 1, and the data are mean values ± SD ( n = 4). For statistical analysis, the mutants were compared with the WT using the student's t ‐test: *, p

    Journal: Environmental Microbiology

    Article Title: Set1 and Kdm5 are antagonists for H3K4 methylation and regulators of the major conidiation‐specific transcription factor gene ABA1 in Fusarium fujikuroi

    doi: 10.1111/1462-2920.14339

    Figure Lengend Snippet: Relative expression as well as H3K4me3 levels of conidiation‐related TF genes in Δ set1 and Δ kdm5 mutants. Indicated strains were grown on V8 agar in the presence of a 12 h light/12 h dark cycle for 3 days to induce conidiation. A . RNA was extracted and the relative expression of ABA1 , FLB3 and FLB4 was determined by RT‐qPCR. B . ChIP‐qPCR was performed for the same genes using the H3K4me3 antibody. Therefore, the enriched samples (precipitated by antibody) were normalized to the respective input samples (initially applied chromatin). The WT was arbitrarily set to 1, and the data are mean values ± SD ( n = 4). For statistical analysis, the mutants were compared with the WT using the student's t ‐test: *, p

    Article Snippet: Expression analysis via northern blot and qPCR RNA from lyophilised and ground mycelium was extracted with the TRI Reagent (Sigma‐Aldrich, Steinheim, Germany).

    Techniques: Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Regulatory network of conidiation. A,B . The strains were grown on V8 agar in the presence of a 12 h light/12 h dark cycle for 3 days. Then, RNA was extracted and the relative expression of ABA1 and WET1 was determined by RT‐qPCR. Data are mean values ( n = 3). C . Indicated strains were grown for 14 days under above described conditions prior to analysis of conidia formation. Data are mean values ± SD ( n = 4). For statistical analysis, the mutants were compared with the WT using the student's t ‐test: *, p

    Journal: Environmental Microbiology

    Article Title: Set1 and Kdm5 are antagonists for H3K4 methylation and regulators of the major conidiation‐specific transcription factor gene ABA1 in Fusarium fujikuroi

    doi: 10.1111/1462-2920.14339

    Figure Lengend Snippet: Regulatory network of conidiation. A,B . The strains were grown on V8 agar in the presence of a 12 h light/12 h dark cycle for 3 days. Then, RNA was extracted and the relative expression of ABA1 and WET1 was determined by RT‐qPCR. Data are mean values ( n = 3). C . Indicated strains were grown for 14 days under above described conditions prior to analysis of conidia formation. Data are mean values ± SD ( n = 4). For statistical analysis, the mutants were compared with the WT using the student's t ‐test: *, p

    Article Snippet: Expression analysis via northern blot and qPCR RNA from lyophilised and ground mycelium was extracted with the TRI Reagent (Sigma‐Aldrich, Steinheim, Germany).

    Techniques: Expressing, Quantitative RT-PCR

    RT-qPCR detection of GRSPaV from total RNA isolated from grapevine leaves. Total RNA was isolated from V. vinifera var. Chardonnay using Spectrum™ Plant Total RNA kit (Sigma), Plant/fungi total RNA kit (Norgen) and AccuPrep viral RNA extraction kit (Bioneer). cDNA prepared with oligo d(T) on 2 μg of total RNA were subjected to SYBR Green quantitative real-time PCR with primers targeting GRSPaV capsid protein gene, grape actin1 and the gene encoding ubiquitin-60S ribosomal protein L40-2 (Additional file 1 : Table S1). Shown is the amplification plot ( a ) and melt curve ( b ) from cDNAs prepared from RNAs isolated with Sigma (blue), Norgen (purple) and Bioneer (green). The C q and melting temperature (T m ) of two technical replicates for all the samples and genes are given in the table below

    Journal: Virology Journal

    Article Title: A highly effective and versatile technology for the isolation of RNAs from grapevines and other woody perennials for use in virus diagnostics

    doi: 10.1186/s12985-015-0376-3

    Figure Lengend Snippet: RT-qPCR detection of GRSPaV from total RNA isolated from grapevine leaves. Total RNA was isolated from V. vinifera var. Chardonnay using Spectrum™ Plant Total RNA kit (Sigma), Plant/fungi total RNA kit (Norgen) and AccuPrep viral RNA extraction kit (Bioneer). cDNA prepared with oligo d(T) on 2 μg of total RNA were subjected to SYBR Green quantitative real-time PCR with primers targeting GRSPaV capsid protein gene, grape actin1 and the gene encoding ubiquitin-60S ribosomal protein L40-2 (Additional file 1 : Table S1). Shown is the amplification plot ( a ) and melt curve ( b ) from cDNAs prepared from RNAs isolated with Sigma (blue), Norgen (purple) and Bioneer (green). The C q and melting temperature (T m ) of two technical replicates for all the samples and genes are given in the table below

    Article Snippet: RT-PCR and RT-qPCR Total RNA from grape leaves isolated with kits from Sigma, Bioneer and Norgen were used in RT-PCR and RT-qPCR.

    Techniques: Quantitative RT-PCR, Isolation, RNA Extraction, SYBR Green Assay, Real-time Polymerase Chain Reaction, Amplification

    The AGE-RAGE pathway triggers autophagic death of cardiomyocytes. (A) Viability of NRVMs was assessed by the CCK8 assay after treatment with various concentrations of AGE. (B) Cell viability measured by the CCK8 assay after AGE treatment (400 μg/ml) in NRVMs with RAGE overexpression or knockdown. (C) Expression of RAGE and Beclin 1 were detected by western blotting after AGE treatment of NRVMs with RAGE overexpression or knockdown. Representative figures are from different blots in Supplementary Figure S1 . (D,E) Quantitative analysis of RAGE and Beclin 1 protein levels. (F,G) NRVMs were infected with short hairpin RNA (shRNA) targeting ATG5, ATG7, or the scramble control. Expression of ATG5 and ATG7 was detected by q-RT-PCR. (H) Knockdown of ATG5 reduced the AGE-induced death of NRVMs. n = 3. Data are the mean ± SE., ∗ P

    Journal: Frontiers in Physiology

    Article Title: Inhibiting Receptor of Advanced Glycation End Products Attenuates Pressure Overload-Induced Cardiac Dysfunction by Preventing Excessive Autophagy

    doi: 10.3389/fphys.2018.01333

    Figure Lengend Snippet: The AGE-RAGE pathway triggers autophagic death of cardiomyocytes. (A) Viability of NRVMs was assessed by the CCK8 assay after treatment with various concentrations of AGE. (B) Cell viability measured by the CCK8 assay after AGE treatment (400 μg/ml) in NRVMs with RAGE overexpression or knockdown. (C) Expression of RAGE and Beclin 1 were detected by western blotting after AGE treatment of NRVMs with RAGE overexpression or knockdown. Representative figures are from different blots in Supplementary Figure S1 . (D,E) Quantitative analysis of RAGE and Beclin 1 protein levels. (F,G) NRVMs were infected with short hairpin RNA (shRNA) targeting ATG5, ATG7, or the scramble control. Expression of ATG5 and ATG7 was detected by q-RT-PCR. (H) Knockdown of ATG5 reduced the AGE-induced death of NRVMs. n = 3. Data are the mean ± SE., ∗ P

    Article Snippet: Quantitative Real-Time PCR Total RNA was extracted from LV tissue by using an RNAprep Pure Tissue Kit (Tiangen, Beijing, China), after which first-strand cDNA was synthesized with a FastKing RT Kit (Tiangen, Beijing, China) and quantitative PCR (qPCR) was performed using SYBR® Premix Ex TaqTM II (Takara, Beijing, China).

    Techniques: CCK-8 Assay, Over Expression, Expressing, Western Blot, Infection, shRNA, Reverse Transcription Polymerase Chain Reaction

    MiR‐24‐3p and MEN1 act in a negative feedback model. (a–d) Mammary epithelial cells were transfected with miR‐24‐3p mimics/mimics NC (mimics/mimics NC; a and c) and/or miR‐24‐3p inhibitor/inhibitor NC (inhibitor/ inhibitor NC; b,d), and cells were harvested for RNA and protein extraction at 24, 48, and 72 hr after transfection. The expression of bovine MEN1 mRNA at 24 hr (a,c) and protein menin in a time course (b,d) were assessed using qRT‐PCR and western blot technology, respectively. Representative WB images of the expression of protein menin at 24, 28, and 72 hr after transfection of indicated miR‐24‐3p are shown. The data are shown as the relative expression levels normalized to the internal control, β‐actin. * p

    Journal: Journal of Cellular Physiology

    Article Title: MiR‐24‐3p regulates cell proliferation and milk protein synthesis of mammary epithelial cells through menin in dairy cows, et al. MiR‐24‐3p regulates cell proliferation and milk protein synthesis of mammary epithelial cells through menin in dairy cows

    doi: 10.1002/jcp.27017

    Figure Lengend Snippet: MiR‐24‐3p and MEN1 act in a negative feedback model. (a–d) Mammary epithelial cells were transfected with miR‐24‐3p mimics/mimics NC (mimics/mimics NC; a and c) and/or miR‐24‐3p inhibitor/inhibitor NC (inhibitor/ inhibitor NC; b,d), and cells were harvested for RNA and protein extraction at 24, 48, and 72 hr after transfection. The expression of bovine MEN1 mRNA at 24 hr (a,c) and protein menin in a time course (b,d) were assessed using qRT‐PCR and western blot technology, respectively. Representative WB images of the expression of protein menin at 24, 28, and 72 hr after transfection of indicated miR‐24‐3p are shown. The data are shown as the relative expression levels normalized to the internal control, β‐actin. * p

    Article Snippet: 2.3 Quantitative RT‐PCR Total RNA was isolated from transfected MAC‐T cells and/or bovine mammary gland tissue using the miRcute miRNA Isolation Kit (TIANGEN, Beijing, China) in accordance with the manufacturer's instructions.

    Techniques: Activated Clotting Time Assay, Transfection, Protein Extraction, Expressing, Quantitative RT-PCR, Western Blot

    ETV5 and SOX9 do not regulate N-cadherin ( A ) Transcriptional regulators deregulated in the presence of AR-V7 compared to AR-WT with known binding sites in CDH2 gene are listed. ( B ) IPA analysis from deregulated genes in the presence of AR-V7 highlighted a network in which ETV5 was a direct regulator of CDH2. Green: downregulated expression; Red: upregulated expression. ( C ) A lentiviral inducible system was used to verify ETV5 expression in the presence of AR variants observed in our RNA-seq data. AR-WT and AR variants expression were induced with 20 ng/mL doxycycline. LNCaP expressing AR-WT and AR variants were cultured in the presence of 10nM DHT and ETV5 mRNA expression level was analyzed by real-time PCR 4 days after induction. ( D ) To analyze the impact of ETV5 on N-cadherin expression, AR-WT and AR-V7 were induced in LNCaP with 20 ng/mL doxycycline and cells were transfected with 50 nM of siRNA against ETV5 (left panel). After 48 h, total mRNA was extracted and CDH2 mRNA expression level was assessed by qRT-PCR (right panel). For all qRT-PCR analyses, the results were normalized to β-ACTIN. Relative expression is represented as the mean of ΔΔCt ± SEM of three independent experiments. NS: not significant, *** P

    Journal: Oncotarget

    Article Title: Dual effects of constitutively active androgen receptor and full-length androgen receptor for N-cadherin regulation in prostate cancer

    doi: 10.18632/oncotarget.18270

    Figure Lengend Snippet: ETV5 and SOX9 do not regulate N-cadherin ( A ) Transcriptional regulators deregulated in the presence of AR-V7 compared to AR-WT with known binding sites in CDH2 gene are listed. ( B ) IPA analysis from deregulated genes in the presence of AR-V7 highlighted a network in which ETV5 was a direct regulator of CDH2. Green: downregulated expression; Red: upregulated expression. ( C ) A lentiviral inducible system was used to verify ETV5 expression in the presence of AR variants observed in our RNA-seq data. AR-WT and AR variants expression were induced with 20 ng/mL doxycycline. LNCaP expressing AR-WT and AR variants were cultured in the presence of 10nM DHT and ETV5 mRNA expression level was analyzed by real-time PCR 4 days after induction. ( D ) To analyze the impact of ETV5 on N-cadherin expression, AR-WT and AR-V7 were induced in LNCaP with 20 ng/mL doxycycline and cells were transfected with 50 nM of siRNA against ETV5 (left panel). After 48 h, total mRNA was extracted and CDH2 mRNA expression level was assessed by qRT-PCR (right panel). For all qRT-PCR analyses, the results were normalized to β-ACTIN. Relative expression is represented as the mean of ΔΔCt ± SEM of three independent experiments. NS: not significant, *** P

    Article Snippet: Quantitative real-time PCR Total RNA was isolated using NucleoSpin® RNA II assay (Macherey-Nagel) according to the manufacturer's procedure and 400 ng of total RNA were reverse transcribed using iScript kit (Bio-Rad).

    Techniques: Binding Assay, Indirect Immunoperoxidase Assay, Expressing, RNA Sequencing Assay, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Quantitative RT-PCR

    Expression of α1-adrenergic receptors in cortical and subcortical brain arterioles. ( A ) Western blot analysis was performed for cortical and subcortical brain arterioles from 14 sheep, as described in the methods section. α1-adrenergic receptors (α1-AR) are detectable in cortex (Cx) and subcortex (Scx). 1–4, samples from different sheep; AU, arbitrary units; Ref., reference sample; ( B ) Quantification of receptor expression after normalization of band intensities to the housekeeping protein β-actin shows no differences of expression levels in cortex and subcortex, respectively; ( C ) Quantitative PCR-analysis was done using cDNA reverse transcribed from total RNA of 7 sheep. PCR-products of correct size were consistently detected for housekeeping genes GAPDH and ZO-1 , and for the α1-adrenergic receptors A ( ADRA1A ) and D ( ADRA1D ), but not for the B-subtype receptor ( ADRA1B ). A, B, depict examples from two individual sheep; H 2 O, no cDNA control; ( D ) Quantitation of receptor mRNA-expression in relation to the GAPDH mRNA and the ZO-1 mRNA , respectively, by the ΔΔ C t -method. As some data sets were not normally distributed all quantitations are presented as box plots, where boxes represent 25th and 75th percentiles, respectively. Medians are indicated by horizontal lines. Whiskers indicate 10th and 90th percentiles, respectively. p -values indicating significant differences are given in the panels.

    Journal: International Journal of Molecular Sciences

    Article Title: Redistribution of Cerebral Blood Flow during Severe Hypovolemia and Reperfusion in a Sheep Model: Critical Role of α1-Adrenergic Signaling

    doi: 10.3390/ijms18051031

    Figure Lengend Snippet: Expression of α1-adrenergic receptors in cortical and subcortical brain arterioles. ( A ) Western blot analysis was performed for cortical and subcortical brain arterioles from 14 sheep, as described in the methods section. α1-adrenergic receptors (α1-AR) are detectable in cortex (Cx) and subcortex (Scx). 1–4, samples from different sheep; AU, arbitrary units; Ref., reference sample; ( B ) Quantification of receptor expression after normalization of band intensities to the housekeeping protein β-actin shows no differences of expression levels in cortex and subcortex, respectively; ( C ) Quantitative PCR-analysis was done using cDNA reverse transcribed from total RNA of 7 sheep. PCR-products of correct size were consistently detected for housekeeping genes GAPDH and ZO-1 , and for the α1-adrenergic receptors A ( ADRA1A ) and D ( ADRA1D ), but not for the B-subtype receptor ( ADRA1B ). A, B, depict examples from two individual sheep; H 2 O, no cDNA control; ( D ) Quantitation of receptor mRNA-expression in relation to the GAPDH mRNA and the ZO-1 mRNA , respectively, by the ΔΔ C t -method. As some data sets were not normally distributed all quantitations are presented as box plots, where boxes represent 25th and 75th percentiles, respectively. Medians are indicated by horizontal lines. Whiskers indicate 10th and 90th percentiles, respectively. p -values indicating significant differences are given in the panels.

    Article Snippet: Quantitative RT-PCR Total RNA was extracted from cortex and thalamus of 8 sheep using NucleoSpin RNA (Macherey-Nagel, Düren, Germany), and reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (AppliedBiosystems, Darmstadt, Germany). qRT-PCR was performed in a StepOnePlus cycler (Applied Biosystems) using GoTaq qPCR Master Mix (Promega, Mannheim, Germany), and the following primers (Gene symbol, forward primer, reverse primer): ADRA1A , 5′-ACTACATCGTCAACCTGGCG-3′, 5′-GGTAGCGCAGAGGATAGCTC-3′; ADRA1B , 5′-CCTTCAAGCTCTTGCCCGA-3′, 5′-CCAGGGGCATGTTGCTTTG-3′; ADRA1D , 5′-CATTGTCGTGGGCGTCTTTG-3′, 5′-TGTTGAAGTAGCCCAGCCAG-3′; GAPDH , 5′-GAAGGTCGGAGTGAACGGAT-3′, 5′-GATGACGAGCTTCCCGTTCT-3′; and ZO1 , 5′-CTCCAGGCCCTTACCTTTCG-3′, 5′-CTCGTAAAGAGTCGGCGTGT-3′.

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Quantitation Assay

    Depletion of lincRNAs increases MAF mRNA levels in HEK293 cells. HEK293 cells were reverse-transfected with a control siRNA, 5 nM MAFTRR siRNA (A–C) or 5 nM LINC01229 siRNA (D–F) , and quantitative PCR was performed on RNA collected at 48 h post-transfection. (A) MAFTRR RNA transcript levels. (B) MAF transcript levels. (C) LINC01229 RNA transcript levels. (D) LINC01229 RNA transcript levels. (E) MAF RNA transcript levels. (F) MAFTRR transcript levels. Data represent four biological replicates; statistical significance was determined using unpaired t -test with one tail. Error bars denote standard error of the mean and asterisks indicate significance: ∗ p

    Journal: Frontiers in Genetics

    Article Title: Functional Urate-Associated Genetic Variants Influence Expression of lincRNAs LINC01229 and MAFTRR

    doi: 10.3389/fgene.2018.00733

    Figure Lengend Snippet: Depletion of lincRNAs increases MAF mRNA levels in HEK293 cells. HEK293 cells were reverse-transfected with a control siRNA, 5 nM MAFTRR siRNA (A–C) or 5 nM LINC01229 siRNA (D–F) , and quantitative PCR was performed on RNA collected at 48 h post-transfection. (A) MAFTRR RNA transcript levels. (B) MAF transcript levels. (C) LINC01229 RNA transcript levels. (D) LINC01229 RNA transcript levels. (E) MAF RNA transcript levels. (F) MAFTRR transcript levels. Data represent four biological replicates; statistical significance was determined using unpaired t -test with one tail. Error bars denote standard error of the mean and asterisks indicate significance: ∗ p

    Article Snippet: Quantitative PCR Total RNA was isolated from control and siRNA-treated HEK293 cells at 48 h post-treatment using the NucleoSpin RNA kit (Macherey-Nagel). cDNA was synthesized with qScript cDNA SuperMix (Quanta Biosciences).

    Techniques: Transfection, Real-time Polymerase Chain Reaction

    Virus shedding in Syrian hamsters inoculated with Nipah virus via different routes. Groups of eight hamsters were inoculated with 10 7 TCID 50 of Nipah virus (strain Bangladesh/200401066) intranasally (red bars) or esophageally (blue bars) and with 5×10 8 TCID 50 via drinking (green bars) and nasal (A), pharyngeal (B), urogenital (C) and rectal (D) swabs were collected daily until 12 dpi. Viral load in the swabs was determined as TCID 50 equivalents by real-time RT-PCR. TCID 50 equivalents were extrapolated from standard curves generated by adding dilutions of RNA extracted from a Nipah virus stock with a known virus titer in parallel to each run. Geometric mean viral loads are displayed; error bars indicate standard deviation.

    Journal: PLoS Pathogens

    Article Title: Foodborne Transmission of Nipah Virus in Syrian Hamsters

    doi: 10.1371/journal.ppat.1004001

    Figure Lengend Snippet: Virus shedding in Syrian hamsters inoculated with Nipah virus via different routes. Groups of eight hamsters were inoculated with 10 7 TCID 50 of Nipah virus (strain Bangladesh/200401066) intranasally (red bars) or esophageally (blue bars) and with 5×10 8 TCID 50 via drinking (green bars) and nasal (A), pharyngeal (B), urogenital (C) and rectal (D) swabs were collected daily until 12 dpi. Viral load in the swabs was determined as TCID 50 equivalents by real-time RT-PCR. TCID 50 equivalents were extrapolated from standard curves generated by adding dilutions of RNA extracted from a Nipah virus stock with a known virus titer in parallel to each run. Geometric mean viral loads are displayed; error bars indicate standard deviation.

    Article Snippet: Quantitative PCR RNA was extracted from swab samples using the NucleoSpin 96 Virus Core kit (Macherey-Nagel) and a Corbett Robotics model CAS 1820 automatic RNA extractor.

    Techniques: Quantitative RT-PCR, Generated, Standard Deviation

    Transmission of Nipah virus (strain Bangladesh/200401066) in Syrian hamsters. Shedding of Nipah virus in inoculated (left panels) and naïve (right panels) animals in fomite, contact and aerosol transmission. Hamsters were inoculated intranasally with 10 7 TCID 50 of Nipah virus (strain Bangladesh/200401066) or via drinking of 5×10 8 TCID 50 of Nipah virus (strain Bangladesh/200401066) in artificial palm sap; nasal (white bars) and pharyngeal (black bars) swabs were collected daily. Seroconversion in naïve hamsters at 28 dpi as determined by ELISA is indicated on the right as the number of seroconverted hamsters/total number of exposed hamsters. Viral load in the swabs was determined as TCID 50 equivalents by real-time RT-PCR. TCID 50 equivalents were extrapolated from standard curves generated by adding dilutions of RNA extracted from a Nipah virus stock with a known virus titer in parallel to each run. Geometric mean viral loads are displayed; error bars indicate standard deviation.

    Journal: PLoS Pathogens

    Article Title: Foodborne Transmission of Nipah Virus in Syrian Hamsters

    doi: 10.1371/journal.ppat.1004001

    Figure Lengend Snippet: Transmission of Nipah virus (strain Bangladesh/200401066) in Syrian hamsters. Shedding of Nipah virus in inoculated (left panels) and naïve (right panels) animals in fomite, contact and aerosol transmission. Hamsters were inoculated intranasally with 10 7 TCID 50 of Nipah virus (strain Bangladesh/200401066) or via drinking of 5×10 8 TCID 50 of Nipah virus (strain Bangladesh/200401066) in artificial palm sap; nasal (white bars) and pharyngeal (black bars) swabs were collected daily. Seroconversion in naïve hamsters at 28 dpi as determined by ELISA is indicated on the right as the number of seroconverted hamsters/total number of exposed hamsters. Viral load in the swabs was determined as TCID 50 equivalents by real-time RT-PCR. TCID 50 equivalents were extrapolated from standard curves generated by adding dilutions of RNA extracted from a Nipah virus stock with a known virus titer in parallel to each run. Geometric mean viral loads are displayed; error bars indicate standard deviation.

    Article Snippet: Quantitative PCR RNA was extracted from swab samples using the NucleoSpin 96 Virus Core kit (Macherey-Nagel) and a Corbett Robotics model CAS 1820 automatic RNA extractor.

    Techniques: Transmission Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Generated, Standard Deviation

    Adipose-derived mesenchymal stem cells have higher levels of DNA damage-related p53 targets, similar levels of Bcl-2 family p53 targets, and a lower p16 and p19 expression than bone marrow mesenchymal stem cells. Independent nine to 10 preparations (A, C, D, I, J) or six preparations (B, E, F, G, H) of paired adipose-derived mesenchymal stem cells (ASCs) and bone marrow mesenchymal stem cells (BM-MSCs; prepared from the same male or female mouse) were analyzed for RNA levels of the indicated gene by quantitative real-time PCR. * P

    Journal: Stem Cell Research & Therapy

    Article Title: Relative genomic stability of adipose tissue derived mesenchymal stem cells: analysis of ploidy, H19 long non-coding RNA and p53 activity

    doi: 10.1186/scrt529

    Figure Lengend Snippet: Adipose-derived mesenchymal stem cells have higher levels of DNA damage-related p53 targets, similar levels of Bcl-2 family p53 targets, and a lower p16 and p19 expression than bone marrow mesenchymal stem cells. Independent nine to 10 preparations (A, C, D, I, J) or six preparations (B, E, F, G, H) of paired adipose-derived mesenchymal stem cells (ASCs) and bone marrow mesenchymal stem cells (BM-MSCs; prepared from the same male or female mouse) were analyzed for RNA levels of the indicated gene by quantitative real-time PCR. * P

    Article Snippet: Quantitative real-time PCR RNA from passage 4 ASCs and passage 7 or higher BM MSCs was extracted using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany), and cDNA was prepared using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s protocols.

    Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction

    Independent adipose-derived mesenchymal stem cell preparations retain their diploid state under hypoxia and react homogeneously to changing oxygen conditions. Independent preparations of paired (A) adipose-derived mesenchymal stem cells (ASCs) and (B) bone marrow mesenchymal stem cells (BM MSCs; prepared from the same male or female mouse) were expanded in Mesencult under hypoxic conditions (3% oxygen) and their DNA content was analyzed by flow cytometry. Resulting plots from male/female adipose-derived mesenchymal stem cells/BM MSCs were overlaid and are presented in a single graph, diploid (2N) and tetraploid (4N); six female and six male adipose-derived mesenchymal stem cells and six total female and male BM MSCs were used. (C) Graph summarizing the percentage of adipose-derived mesenchymal stem cells and BM MSCs that became polyploid out of all cell preparations cultured during the study. (D) , (E) (F) Comparison of the RNA level of independently derived adipose-derived mesenchymal stem cells or BM MSCs that were expanded either in hypoxic (H) or normoxic (N; normal oxygen) conditions made by quantitative real-time PCR. * P

    Journal: Stem Cell Research & Therapy

    Article Title: Relative genomic stability of adipose tissue derived mesenchymal stem cells: analysis of ploidy, H19 long non-coding RNA and p53 activity

    doi: 10.1186/scrt529

    Figure Lengend Snippet: Independent adipose-derived mesenchymal stem cell preparations retain their diploid state under hypoxia and react homogeneously to changing oxygen conditions. Independent preparations of paired (A) adipose-derived mesenchymal stem cells (ASCs) and (B) bone marrow mesenchymal stem cells (BM MSCs; prepared from the same male or female mouse) were expanded in Mesencult under hypoxic conditions (3% oxygen) and their DNA content was analyzed by flow cytometry. Resulting plots from male/female adipose-derived mesenchymal stem cells/BM MSCs were overlaid and are presented in a single graph, diploid (2N) and tetraploid (4N); six female and six male adipose-derived mesenchymal stem cells and six total female and male BM MSCs were used. (C) Graph summarizing the percentage of adipose-derived mesenchymal stem cells and BM MSCs that became polyploid out of all cell preparations cultured during the study. (D) , (E) (F) Comparison of the RNA level of independently derived adipose-derived mesenchymal stem cells or BM MSCs that were expanded either in hypoxic (H) or normoxic (N; normal oxygen) conditions made by quantitative real-time PCR. * P

    Article Snippet: Quantitative real-time PCR RNA from passage 4 ASCs and passage 7 or higher BM MSCs was extracted using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany), and cDNA was prepared using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s protocols.

    Techniques: Derivative Assay, Flow Cytometry, Cytometry, Cell Culture, Real-time Polymerase Chain Reaction

    DNMT3B modulates exon 4–5–6 skipping by physically interacting with hnRNP-LL and CD45 pre-mRNA. ( A ) Native RNA immunoprecipitation assay (RIP-qPCR) shows DNMT3B interaction with CD45 pre-mRNA in control and ICF1 samples. Binding to HOXC4 mRNA is reported as negative control; ( B ) DNMT3B enrichment at DNA (exons 4, 5 and 6) of CD45 gene by ChIP assay; ( C and D ) Expression level of hnRNP-LL in ICF1 samples and controls by qPCR and western blot, respectively; ( E ) CpG methylation level [log2 (methylated/unmethylated)] at hnRNP-LL promoter in ICF1 samples; ( F ) DNMT3B and hnRNP-LL physically interact in ICF1p2 sample as shown in co-immunoprecipitation experiments; ( G ) Expression level (qPCR) of CD45RABC and the constitutive exon9 using isoform specific oligonucleotides upon siDNMT3B and control siRNAs transfection; ( H ) Semi-quantitative PCR amplification of CD45 splicing isoforms in overexpressing-hnRNP-LL ICF1p2 sample upon siDNMT3B and control siRNAs transfection. * P -adj

    Journal: Nucleic Acids Research

    Article Title: ICF-specific DNMT3B dysfunction interferes with intragenic regulation of mRNA transcription and alternative splicing

    doi: 10.1093/nar/gkx163

    Figure Lengend Snippet: DNMT3B modulates exon 4–5–6 skipping by physically interacting with hnRNP-LL and CD45 pre-mRNA. ( A ) Native RNA immunoprecipitation assay (RIP-qPCR) shows DNMT3B interaction with CD45 pre-mRNA in control and ICF1 samples. Binding to HOXC4 mRNA is reported as negative control; ( B ) DNMT3B enrichment at DNA (exons 4, 5 and 6) of CD45 gene by ChIP assay; ( C and D ) Expression level of hnRNP-LL in ICF1 samples and controls by qPCR and western blot, respectively; ( E ) CpG methylation level [log2 (methylated/unmethylated)] at hnRNP-LL promoter in ICF1 samples; ( F ) DNMT3B and hnRNP-LL physically interact in ICF1p2 sample as shown in co-immunoprecipitation experiments; ( G ) Expression level (qPCR) of CD45RABC and the constitutive exon9 using isoform specific oligonucleotides upon siDNMT3B and control siRNAs transfection; ( H ) Semi-quantitative PCR amplification of CD45 splicing isoforms in overexpressing-hnRNP-LL ICF1p2 sample upon siDNMT3B and control siRNAs transfection. * P -adj

    Article Snippet: Quantitative real time PCR Total RNA from B-LCLs was reverse-transcribed using iScript cDNA Synthesis kit (Bio-Rad San Diego, CA, USA).

    Techniques: Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Negative Control, Chromatin Immunoprecipitation, Expressing, Western Blot, CpG Methylation Assay, Methylation, Transfection, Amplification

    IL-1R1 and IRF4 are required for human Th17 cell differentiation . (A) IRF4 gene expression in the CD4 + T cell from RR MS patients is significantly increased in comparison to HCs. CD4 + T cells derived from six RR MS patients and six HCs were separated using magnetic beads, and the total RNA was extracted. The gene expression of IRF4 and 18S was measured by RT-PCR. The results are expressed as relative gene expression normalized for 18S mRNA expression. Statistical analysis was performed using t -tests. * p

    Journal: Frontiers in Immunology

    Article Title: Activated IL-1RI Signaling Pathway Induces Th17 Cell Differentiation via Interferon Regulatory Factor 4 Signaling in Patients with Relapsing-Remitting Multiple Sclerosis

    doi: 10.3389/fimmu.2016.00543

    Figure Lengend Snippet: IL-1R1 and IRF4 are required for human Th17 cell differentiation . (A) IRF4 gene expression in the CD4 + T cell from RR MS patients is significantly increased in comparison to HCs. CD4 + T cells derived from six RR MS patients and six HCs were separated using magnetic beads, and the total RNA was extracted. The gene expression of IRF4 and 18S was measured by RT-PCR. The results are expressed as relative gene expression normalized for 18S mRNA expression. Statistical analysis was performed using t -tests. * p

    Article Snippet: Quantitative RT-PCR Total RNA was isolated from CD4+ , CD4+ CD45RA+ , CD4+ CD45RO+ T cells and reverse-transcribed to cDNA using an iSCRIPT cDNA synthesis kit (Bio-Rad).

    Techniques: Cell Differentiation, Expressing, Mass Spectrometry, Derivative Assay, Magnetic Beads, Reverse Transcription Polymerase Chain Reaction

    IL-1R1 expression is higher in the in vitro -differentiated Th17 cells in comparison to Th1 and Th2 cultures . CD4 + CD45RA + cells were derived from three HCs and stimulated with plate-immobilized anti-CD3 and anti-CD28 mAb in a serum-free medium in the absence or presence of Th1, Th2, or Th17-polarizing cytokines. After 72 h, total RNA was extracted and gene expression of IL-1R1 and 18S was measured by RT-PCR. The results are expressed as relative gene expression normalized for 18S mRNA expression. Statistical analysis was performed using ANOVA. *** p

    Journal: Frontiers in Immunology

    Article Title: Activated IL-1RI Signaling Pathway Induces Th17 Cell Differentiation via Interferon Regulatory Factor 4 Signaling in Patients with Relapsing-Remitting Multiple Sclerosis

    doi: 10.3389/fimmu.2016.00543

    Figure Lengend Snippet: IL-1R1 expression is higher in the in vitro -differentiated Th17 cells in comparison to Th1 and Th2 cultures . CD4 + CD45RA + cells were derived from three HCs and stimulated with plate-immobilized anti-CD3 and anti-CD28 mAb in a serum-free medium in the absence or presence of Th1, Th2, or Th17-polarizing cytokines. After 72 h, total RNA was extracted and gene expression of IL-1R1 and 18S was measured by RT-PCR. The results are expressed as relative gene expression normalized for 18S mRNA expression. Statistical analysis was performed using ANOVA. *** p

    Article Snippet: Quantitative RT-PCR Total RNA was isolated from CD4+ , CD4+ CD45RA+ , CD4+ CD45RO+ T cells and reverse-transcribed to cDNA using an iSCRIPT cDNA synthesis kit (Bio-Rad).

    Techniques: Expressing, In Vitro, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

    IL-1R1 signaling induces Th17 cell differentiation . CD4 + CD45RA + cells were derived from three HCs (A) and seven RR MS patients (B) and transfected with a control siRNA A or siRNA IL-1R1, stimulated with plate-immobilized anti-CD3 and anti-CD28 mAb and cultured in serum-free medium in the absence or presence of Th17-polarizing cytokines. The total RNA was extracted at 72 h, and the expression of the indicated genes was measured using RT-PCR. The results are expressed as relative gene expression normalized against 18S mRNA expression. Statistical analysis was performed using repeated measures ANOVA, * p

    Journal: Frontiers in Immunology

    Article Title: Activated IL-1RI Signaling Pathway Induces Th17 Cell Differentiation via Interferon Regulatory Factor 4 Signaling in Patients with Relapsing-Remitting Multiple Sclerosis

    doi: 10.3389/fimmu.2016.00543

    Figure Lengend Snippet: IL-1R1 signaling induces Th17 cell differentiation . CD4 + CD45RA + cells were derived from three HCs (A) and seven RR MS patients (B) and transfected with a control siRNA A or siRNA IL-1R1, stimulated with plate-immobilized anti-CD3 and anti-CD28 mAb and cultured in serum-free medium in the absence or presence of Th17-polarizing cytokines. The total RNA was extracted at 72 h, and the expression of the indicated genes was measured using RT-PCR. The results are expressed as relative gene expression normalized against 18S mRNA expression. Statistical analysis was performed using repeated measures ANOVA, * p

    Article Snippet: Quantitative RT-PCR Total RNA was isolated from CD4+ , CD4+ CD45RA+ , CD4+ CD45RO+ T cells and reverse-transcribed to cDNA using an iSCRIPT cDNA synthesis kit (Bio-Rad).

    Techniques: Cell Differentiation, Derivative Assay, Mass Spectrometry, Transfection, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction

    IL-1R1 positively regulates human Th17 cell differentiation in an IRF4-dependent manner . CD4 + CD45RA + cells from six RR MS patients transfected with a control siRNA A or siRNA IRF4 were stimulated with plate-immobilized anti-CD3 and anti-CD28 mAb and cultured in serum-free medium in the absence or presence of Th17-polarizing cytokines. After 72 h, the total RNA was extracted. The gene expression was measured by RT-PCR. The results are expressed as relative gene expression normalized for 18S mRNA expression. Statistical analysis was performed using ANOVA. * p

    Journal: Frontiers in Immunology

    Article Title: Activated IL-1RI Signaling Pathway Induces Th17 Cell Differentiation via Interferon Regulatory Factor 4 Signaling in Patients with Relapsing-Remitting Multiple Sclerosis

    doi: 10.3389/fimmu.2016.00543

    Figure Lengend Snippet: IL-1R1 positively regulates human Th17 cell differentiation in an IRF4-dependent manner . CD4 + CD45RA + cells from six RR MS patients transfected with a control siRNA A or siRNA IRF4 were stimulated with plate-immobilized anti-CD3 and anti-CD28 mAb and cultured in serum-free medium in the absence or presence of Th17-polarizing cytokines. After 72 h, the total RNA was extracted. The gene expression was measured by RT-PCR. The results are expressed as relative gene expression normalized for 18S mRNA expression. Statistical analysis was performed using ANOVA. * p

    Article Snippet: Quantitative RT-PCR Total RNA was isolated from CD4+ , CD4+ CD45RA+ , CD4+ CD45RO+ T cells and reverse-transcribed to cDNA using an iSCRIPT cDNA synthesis kit (Bio-Rad).

    Techniques: Cell Differentiation, Mass Spectrometry, Transfection, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction

    IL-1RI gene expression is increased in CD4 + , CD4 + CD45RA + , and CD4 + CD45RO + cells from RR MS patients in comparison to HCs . CD4 + , CD4 + CD45RA + naive, and CD4 + CD45RO + memory T cells derived from six RR MS patients and six HCs were separated using magnetic beads. The total RNA was harvested and the gene expression of IL-1RI and 18S was measured using qRT-PCR. The results are presented as the relative gene expression normalized against 18S mRNA. Statistical analysis was performed using unpaired t -test.

    Journal: Frontiers in Immunology

    Article Title: Activated IL-1RI Signaling Pathway Induces Th17 Cell Differentiation via Interferon Regulatory Factor 4 Signaling in Patients with Relapsing-Remitting Multiple Sclerosis

    doi: 10.3389/fimmu.2016.00543

    Figure Lengend Snippet: IL-1RI gene expression is increased in CD4 + , CD4 + CD45RA + , and CD4 + CD45RO + cells from RR MS patients in comparison to HCs . CD4 + , CD4 + CD45RA + naive, and CD4 + CD45RO + memory T cells derived from six RR MS patients and six HCs were separated using magnetic beads. The total RNA was harvested and the gene expression of IL-1RI and 18S was measured using qRT-PCR. The results are presented as the relative gene expression normalized against 18S mRNA. Statistical analysis was performed using unpaired t -test.

    Article Snippet: Quantitative RT-PCR Total RNA was isolated from CD4+ , CD4+ CD45RA+ , CD4+ CD45RO+ T cells and reverse-transcribed to cDNA using an iSCRIPT cDNA synthesis kit (Bio-Rad).

    Techniques: Expressing, Mass Spectrometry, Derivative Assay, Magnetic Beads, Quantitative RT-PCR

    Co-infection of Gouleako goukovirus (GOLV) and RVFV. (A) C6/36 cells were mock-infected or infected with GOLV (8 MOI) and/or rMP-12 (0.5 MOI) at 28°C. Total RNA was extracted at 72 hpi (left four lanes). Culture supernatants were collected and transferred into either C6/36 cells (middle four lanes) or Vero cells (right four lanes). Total RNA was then extracted at 72 hpi. The presence of L-, M-, and S-segment RNA of GOLV (top panel) or RVFV (bottom panel) was analyzed by Northern blot. (B) Vero or PK15 cells were infected with GOLV (8 MOI) with or without either rMP-12 (0.5 MOI) or rMP12-ΔNSs16/198 (2.0 MOI) at 28°C. Total RNA was extracted at 18 hpi, and the accumulation of viral RNA was analyzed by Northern blot. Top panel: probes to detect negative-sense GOLV L-, M-, and S-segments, Bottom panel: probes to detect negative-sense RVFV L-, M-, and S-segments. GOLV RNA derived from C6/36 cells infected with GOLV (8 MOI) was used for the positive control for GOLV probes on the membrane.

    Journal: PLoS ONE

    Article Title: Risk analysis of inter-species reassortment through a Rift Valley fever phlebovirus MP-12 vaccine strain

    doi: 10.1371/journal.pone.0185194

    Figure Lengend Snippet: Co-infection of Gouleako goukovirus (GOLV) and RVFV. (A) C6/36 cells were mock-infected or infected with GOLV (8 MOI) and/or rMP-12 (0.5 MOI) at 28°C. Total RNA was extracted at 72 hpi (left four lanes). Culture supernatants were collected and transferred into either C6/36 cells (middle four lanes) or Vero cells (right four lanes). Total RNA was then extracted at 72 hpi. The presence of L-, M-, and S-segment RNA of GOLV (top panel) or RVFV (bottom panel) was analyzed by Northern blot. (B) Vero or PK15 cells were infected with GOLV (8 MOI) with or without either rMP-12 (0.5 MOI) or rMP12-ΔNSs16/198 (2.0 MOI) at 28°C. Total RNA was extracted at 18 hpi, and the accumulation of viral RNA was analyzed by Northern blot. Top panel: probes to detect negative-sense GOLV L-, M-, and S-segments, Bottom panel: probes to detect negative-sense RVFV L-, M-, and S-segments. GOLV RNA derived from C6/36 cells infected with GOLV (8 MOI) was used for the positive control for GOLV probes on the membrane.

    Article Snippet: RNA probes for GOLV L-, M-, and S-segments were generated from pSPT18-GOLV-L, pSPT18-GOLV-M, or pSPT18-GOLV-S plasmids via in vitro transcription using the DIG RNA Labeling Kit (Sigma-Aldrich), according to the manufacturer’s instruction.

    Techniques: Infection, Northern Blot, Derivative Assay, Positive Control

    Replication of the M-segment minigenomes of Rift Valley fever phlebovirus (RVFV), Arumowot virus (AMTV), and Gouleako goukovirus (GOLV) in relation to co-expression of RVFV N and L proteins. (A) Schematics of panhandle sequence of M-segments of RVFV, AMTV, and GOLV. (B–D) BHK/T7-9 cells were transfected with plasmids expressing the RVFV-M-rLuc(-) (M-segment minigenome RNA of RVFV) (B), AMTV-M-rLuc(-) (M-segment minigenome RNA of AMTV) (C), or GOLV-M-rLuc(-) (M-segment minigenome RNA of GOLV) (D), and those expressing N and/or L proteins derived from either RVFV or AMTV. The ratio of Renilla luciferase (rLuc) activities to firefly luciferase (fLuc) activities derived from pT7-IRES-fLuc (control plasmid) was shown as percentage: i.e., the same type of minigenome without N and L expression was set as 100%. Bars represent means plus standard errors. Asterisks on error bars represent statistically significant increases compared to samples expressing minigenome only (One-way ANOVA **p

    Journal: PLoS ONE

    Article Title: Risk analysis of inter-species reassortment through a Rift Valley fever phlebovirus MP-12 vaccine strain

    doi: 10.1371/journal.pone.0185194

    Figure Lengend Snippet: Replication of the M-segment minigenomes of Rift Valley fever phlebovirus (RVFV), Arumowot virus (AMTV), and Gouleako goukovirus (GOLV) in relation to co-expression of RVFV N and L proteins. (A) Schematics of panhandle sequence of M-segments of RVFV, AMTV, and GOLV. (B–D) BHK/T7-9 cells were transfected with plasmids expressing the RVFV-M-rLuc(-) (M-segment minigenome RNA of RVFV) (B), AMTV-M-rLuc(-) (M-segment minigenome RNA of AMTV) (C), or GOLV-M-rLuc(-) (M-segment minigenome RNA of GOLV) (D), and those expressing N and/or L proteins derived from either RVFV or AMTV. The ratio of Renilla luciferase (rLuc) activities to firefly luciferase (fLuc) activities derived from pT7-IRES-fLuc (control plasmid) was shown as percentage: i.e., the same type of minigenome without N and L expression was set as 100%. Bars represent means plus standard errors. Asterisks on error bars represent statistically significant increases compared to samples expressing minigenome only (One-way ANOVA **p

    Article Snippet: RNA probes for GOLV L-, M-, and S-segments were generated from pSPT18-GOLV-L, pSPT18-GOLV-M, or pSPT18-GOLV-S plasmids via in vitro transcription using the DIG RNA Labeling Kit (Sigma-Aldrich), according to the manufacturer’s instruction.

    Techniques: Expressing, Sequencing, Transfection, Derivative Assay, Luciferase, Plasmid Preparation

    QPCR custom array heat map of relative gene expression throughout the time course of acute (A) or chronic (B) cuprizone treatment and recovery RNA was isolated from the corpus callosum of mice at each time point shown along the top of the heat map. The time points are indicated as the number of weeks of cuprizone ingestion (0, 3, 6, 9 or 12) followed by the weeks on normal chow for recovery (+3 or +6). Results show fold-change values for each gene from triplicate PCR reactions which were each run on a separate plate. For each gene, relative expression levels are shown with the highest as dark red and the lowest as dark blue. Genes are hierarchically clustered based on correlation with expression of GFAP across each time course. Genes above the arrow on each heat map show a pattern similar to GFAP. The age-matched equivalent to the beginning of cuprizone treatment is shown as 0 week while the equivalent to the end of the time course of treatment and recovery is 20 week ( A ) for the acute and 26 week ( B ) for the chronic time course.

    Journal: ASN NEURO

    Article Title: Astrogliosis during acute and chronic cuprizone demyelination and implications for remyelination

    doi: 10.1042/AN20120062

    Figure Lengend Snippet: QPCR custom array heat map of relative gene expression throughout the time course of acute (A) or chronic (B) cuprizone treatment and recovery RNA was isolated from the corpus callosum of mice at each time point shown along the top of the heat map. The time points are indicated as the number of weeks of cuprizone ingestion (0, 3, 6, 9 or 12) followed by the weeks on normal chow for recovery (+3 or +6). Results show fold-change values for each gene from triplicate PCR reactions which were each run on a separate plate. For each gene, relative expression levels are shown with the highest as dark red and the lowest as dark blue. Genes are hierarchically clustered based on correlation with expression of GFAP across each time course. Genes above the arrow on each heat map show a pattern similar to GFAP. The age-matched equivalent to the beginning of cuprizone treatment is shown as 0 week while the equivalent to the end of the time course of treatment and recovery is 20 week ( A ) for the acute and 26 week ( B ) for the chronic time course.

    Article Snippet: The RNA was purified using the RT2 QPCR-Grade RNA Isolation Kit (SABiosciences, PA-001).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation, Mouse Assay, Polymerase Chain Reaction

    Differential expression of glycogen synthesizing enzymes due to increased (p)ppGpp levels in Δ fumABC . A) STM WT harboring a dual color fluorescence reporter for wraB was cultured in LB o/n and subcultured in minimal medium with or without amino acid (= aa) supplementation (dashed or undashed line, respectively). After 3 h of growth cells were subjected to flow cytometry and sfGFP fluorescence intensity (BL1-H) recorded. Shown is one representative of three independent biological replicates. B) Representative data of WT, Δ fumABC , Δ fumABC Δ glgA and Δ relA Δ spoT strains harboring the wraB reporter grown o/n in LB broth. C) Medians of relative sfGFP fluorescence intensities of strains mentioned in (B). Data were normalized to WT (=1) and represent average values and standard deviation of three biological replicates. D) WT, Δ fumABC and Δ fumABC Δ glgA strains were cultured o/n in LB broth, RNA was extracted and used for cDNA synthesis and consecutive qPCR experiments. 16s rRNA expression levels were used for normalization. Depicted are the expression levels normalized to WT (=1) of glgA and glgC . Shown is one representative assay of three independent biological replicates, consisting each of three technical replicates. Statistical analyses were performed by Student’s t -test and significances are indicated as follows: *, p

    Journal: bioRxiv

    Article Title: Blocks in tricarboxylic acid cycle of Salmonella enterica cause global perturbation of carbon storage, motility and host-pathogen-interaction

    doi: 10.1101/832675

    Figure Lengend Snippet: Differential expression of glycogen synthesizing enzymes due to increased (p)ppGpp levels in Δ fumABC . A) STM WT harboring a dual color fluorescence reporter for wraB was cultured in LB o/n and subcultured in minimal medium with or without amino acid (= aa) supplementation (dashed or undashed line, respectively). After 3 h of growth cells were subjected to flow cytometry and sfGFP fluorescence intensity (BL1-H) recorded. Shown is one representative of three independent biological replicates. B) Representative data of WT, Δ fumABC , Δ fumABC Δ glgA and Δ relA Δ spoT strains harboring the wraB reporter grown o/n in LB broth. C) Medians of relative sfGFP fluorescence intensities of strains mentioned in (B). Data were normalized to WT (=1) and represent average values and standard deviation of three biological replicates. D) WT, Δ fumABC and Δ fumABC Δ glgA strains were cultured o/n in LB broth, RNA was extracted and used for cDNA synthesis and consecutive qPCR experiments. 16s rRNA expression levels were used for normalization. Depicted are the expression levels normalized to WT (=1) of glgA and glgC . Shown is one representative assay of three independent biological replicates, consisting each of three technical replicates. Statistical analyses were performed by Student’s t -test and significances are indicated as follows: *, p

    Article Snippet: qPCR For RNA preparation by ‘hot phenol’ method, bacteria were cultured 18.5 h in LB with aeration.

    Techniques: Expressing, Fluorescence, Cell Culture, Flow Cytometry, Standard Deviation, Real-time Polymerase Chain Reaction

    qPCR analysis of intact (T) and partly-degraded (Tf) RNA T using Transcriptor. Experimental setup and display are as in Fig. 1 . Arrows exemplify the trend of change in Cq values using the highlighted dilution (outlined circles).

    Journal: Scientific Reports

    Article Title: Enzyme- and gene-specific biases in reverse transcription of RNA raise concerns for evaluating gene expression

    doi: 10.1038/s41598-020-65005-0

    Figure Lengend Snippet: qPCR analysis of intact (T) and partly-degraded (Tf) RNA T using Transcriptor. Experimental setup and display are as in Fig. 1 . Arrows exemplify the trend of change in Cq values using the highlighted dilution (outlined circles).

    Article Snippet: To rule out the contribution of potential contaminants due to our RNA extraction techniques, we processed alongside H and T cell line RNAs similar quantities of Stratagene QPCR Human Reference Total RNA (U), which is a high-quality commercial control RNA recommended for quantitative PCR gene-expression analysis.

    Techniques: Real-time Polymerase Chain Reaction

    qPCR analysis of RNA from two cell lines (H and T) using iScript RT performed with either 75 or 600 ng RNA. Differential expression of OAZ1 relative to eEF1A1 is calculated using the ΔΔCq method.

    Journal: Scientific Reports

    Article Title: Enzyme- and gene-specific biases in reverse transcription of RNA raise concerns for evaluating gene expression

    doi: 10.1038/s41598-020-65005-0

    Figure Lengend Snippet: qPCR analysis of RNA from two cell lines (H and T) using iScript RT performed with either 75 or 600 ng RNA. Differential expression of OAZ1 relative to eEF1A1 is calculated using the ΔΔCq method.

    Article Snippet: To rule out the contribution of potential contaminants due to our RNA extraction techniques, we processed alongside H and T cell line RNAs similar quantities of Stratagene QPCR Human Reference Total RNA (U), which is a high-quality commercial control RNA recommended for quantitative PCR gene-expression analysis.

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    A schematic of the experimental design and Cq values of a hypothetical ideal qPCR amplification. A series of 2-fold dilutions of an RNA sample are reverse transcribed (RT), and 2-fold dilutions of each resulting cDNA are analysed by qPCR. Colour intensity of tube (left) and corresponding circle (right) represents predicted amplicon abundances. Samples boxed in blue (left and right) exemplify a set of samples expected to harbour identical Cq values. Relative input quantities are depicted as black steps (right; not to scale). Solid green lines connect samples of one ideal qPCR titration curve (-1 Cq per 2-fold increased input of cDNA in qPCR). Solid pink lines connects the samples of a similar ideal RT titration curve (-1 Cq per 2-fold increased input of RNA in RT).

    Journal: Scientific Reports

    Article Title: Enzyme- and gene-specific biases in reverse transcription of RNA raise concerns for evaluating gene expression

    doi: 10.1038/s41598-020-65005-0

    Figure Lengend Snippet: A schematic of the experimental design and Cq values of a hypothetical ideal qPCR amplification. A series of 2-fold dilutions of an RNA sample are reverse transcribed (RT), and 2-fold dilutions of each resulting cDNA are analysed by qPCR. Colour intensity of tube (left) and corresponding circle (right) represents predicted amplicon abundances. Samples boxed in blue (left and right) exemplify a set of samples expected to harbour identical Cq values. Relative input quantities are depicted as black steps (right; not to scale). Solid green lines connect samples of one ideal qPCR titration curve (-1 Cq per 2-fold increased input of cDNA in qPCR). Solid pink lines connects the samples of a similar ideal RT titration curve (-1 Cq per 2-fold increased input of RNA in RT).

    Article Snippet: To rule out the contribution of potential contaminants due to our RNA extraction techniques, we processed alongside H and T cell line RNAs similar quantities of Stratagene QPCR Human Reference Total RNA (U), which is a high-quality commercial control RNA recommended for quantitative PCR gene-expression analysis.

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Titration

    qPCR analysis of RNA from two cell lines (H and T) as well as a commercial reference (U) with or without fragmentation (fragmented: Hf, Tf and Uf). Experimental setup and display are as in Fig. 1 . Amplicon colour-coding is as in Figs. 2 and 3 . Samples boxed in blue exemplify sets of U1 samples expected to harbour identical Cq values (see Fig. 1 ). Arrows illustrate the shift in Cq of two amplicons upon fragmentation of each of the three tested RNAs.

    Journal: Scientific Reports

    Article Title: Enzyme- and gene-specific biases in reverse transcription of RNA raise concerns for evaluating gene expression

    doi: 10.1038/s41598-020-65005-0

    Figure Lengend Snippet: qPCR analysis of RNA from two cell lines (H and T) as well as a commercial reference (U) with or without fragmentation (fragmented: Hf, Tf and Uf). Experimental setup and display are as in Fig. 1 . Amplicon colour-coding is as in Figs. 2 and 3 . Samples boxed in blue exemplify sets of U1 samples expected to harbour identical Cq values (see Fig. 1 ). Arrows illustrate the shift in Cq of two amplicons upon fragmentation of each of the three tested RNAs.

    Article Snippet: To rule out the contribution of potential contaminants due to our RNA extraction techniques, we processed alongside H and T cell line RNAs similar quantities of Stratagene QPCR Human Reference Total RNA (U), which is a high-quality commercial control RNA recommended for quantitative PCR gene-expression analysis.

    Techniques: Real-time Polymerase Chain Reaction, Amplification

    qPCR analysis of RNA H using two RT kits (iScript and Transcriptor). Experimental setup and display are as in Fig. 1 . Solid lines are average RT titration slopes. For precise values, see text.

    Journal: Scientific Reports

    Article Title: Enzyme- and gene-specific biases in reverse transcription of RNA raise concerns for evaluating gene expression

    doi: 10.1038/s41598-020-65005-0

    Figure Lengend Snippet: qPCR analysis of RNA H using two RT kits (iScript and Transcriptor). Experimental setup and display are as in Fig. 1 . Solid lines are average RT titration slopes. For precise values, see text.

    Article Snippet: To rule out the contribution of potential contaminants due to our RNA extraction techniques, we processed alongside H and T cell line RNAs similar quantities of Stratagene QPCR Human Reference Total RNA (U), which is a high-quality commercial control RNA recommended for quantitative PCR gene-expression analysis.

    Techniques: Real-time Polymerase Chain Reaction, Titration

    Quantitative real-time PCR measuring mRNA expression of decidual marker genes in differentiating HDSC and THESC . Cultures were incubated with cAMP or E2P4 for 3, 6, 9 and 12 days. Cells without stimuli were kept in parallel representing non-stimulated controls (n.c.). RNA extraction and quantitative real-time PCR were performed as described in Methods. For relative quantification of mRNA expression n.c. of day 3 was arbitrarily set at 1 (calibrator). Bars indicate mean values ± SEM of seven (HDSC) and five (THESC) different experiments/PCR reactions performed in duplicates.* depicts p

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Evaluation of human first trimester decidual and telomerase-transformed endometrial stromal cells as model systems of in vitro decidualization

    doi: 10.1186/1477-7827-9-155

    Figure Lengend Snippet: Quantitative real-time PCR measuring mRNA expression of decidual marker genes in differentiating HDSC and THESC . Cultures were incubated with cAMP or E2P4 for 3, 6, 9 and 12 days. Cells without stimuli were kept in parallel representing non-stimulated controls (n.c.). RNA extraction and quantitative real-time PCR were performed as described in Methods. For relative quantification of mRNA expression n.c. of day 3 was arbitrarily set at 1 (calibrator). Bars indicate mean values ± SEM of seven (HDSC) and five (THESC) different experiments/PCR reactions performed in duplicates.* depicts p

    Article Snippet: RNA extraction and quantitative real-time PCR Total RNA was extracted by direct lysis in the culture dishes using TriFast Reagent (PeqLab, Erlangen, D) according to the manufacturer's instructions.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Marker, Incubation, RNA Extraction, Polymerase Chain Reaction

    PACT Ser-246 and Ser-287 are necessary for PKR interaction and downstream inflammatory signaling. ( A ) PACT KO MEFs were reconstituted with FLAG-WT PACT or FLAG-S246A mutant PACT constructs, then treated with 600 mOsm sucrose for 3 hr. Proteins from total cell extracts or co-immunoprecipitated with the FLAG antibody were analyzed via western blot. ( B ) PACT KO MEFs were reconstituted with FLAG-WT PACT, FLAG-S246A, or FLAG-S287A mutant PACT constructs, then treated with 600 mOsm sucrose for 3 hr. Total lysates were analyzed via western blot. ( C ) PACT KO MEFs were reconstituted with FLAG-WT PACT or FLAG-S246A mutant PACT constructs, then treated with 600 mOsm sucrose for 3 hr. RNA was isolated, and Nos2 transcript levels were analyzed via qPCR. ( D ) PKR KO MEFs reconstituted with wild type PKR, PKR mutated at the RNA-binding residues (K64R/K154R), or kinase activity-deficient PKR (K296R) were treated with 500 or 600 mOsm sucrose for the indicated durations. RNA was isolated, and Nos2 transcript levels were analyzed via qPCR.

    Journal: eLife

    Article Title: PACT-mediated PKR activation acts as a hyperosmotic stress intensity sensor weakening osmoadaptation and enhancing inflammation

    doi: 10.7554/eLife.52241

    Figure Lengend Snippet: PACT Ser-246 and Ser-287 are necessary for PKR interaction and downstream inflammatory signaling. ( A ) PACT KO MEFs were reconstituted with FLAG-WT PACT or FLAG-S246A mutant PACT constructs, then treated with 600 mOsm sucrose for 3 hr. Proteins from total cell extracts or co-immunoprecipitated with the FLAG antibody were analyzed via western blot. ( B ) PACT KO MEFs were reconstituted with FLAG-WT PACT, FLAG-S246A, or FLAG-S287A mutant PACT constructs, then treated with 600 mOsm sucrose for 3 hr. Total lysates were analyzed via western blot. ( C ) PACT KO MEFs were reconstituted with FLAG-WT PACT or FLAG-S246A mutant PACT constructs, then treated with 600 mOsm sucrose for 3 hr. RNA was isolated, and Nos2 transcript levels were analyzed via qPCR. ( D ) PKR KO MEFs reconstituted with wild type PKR, PKR mutated at the RNA-binding residues (K64R/K154R), or kinase activity-deficient PKR (K296R) were treated with 500 or 600 mOsm sucrose for the indicated durations. RNA was isolated, and Nos2 transcript levels were analyzed via qPCR.

    Article Snippet: RT-qPCR Total RNA was prepared using TRIzol reagent (Ambion) according to manufacturer’s instructions.

    Techniques: Mutagenesis, Construct, Immunoprecipitation, Western Blot, Isolation, Real-time Polymerase Chain Reaction, RNA Binding Assay, Activity Assay

    NF-κB c-Rel is a novel factor in the adaptive response to hyperosmotic stress. ( A ) Control and shPACT MEFs were treated with 500 or 600 mOsm. Nuclear fractions were isolated, and protein levels were analyzed via western blot. Insert indicates efficiency of PACT depletion from cytoplasmic extracts. ( B ) Control and shc-Rel MEFs were treated with 500 mOsm sucrose. Lysates were assayed for caspase-3 enzymatic activity. ( C ) Control and shc-Rel MEFs were treated with 500 or 600 mOsm sucrose. Nuclear fractions were isolated and protein levels were analyzed via western blot. ( D ) Control and shc-Rel MEFs were treated with 500 or 600 mOsm sucrose. RNA was isolated, and mRNA transcripts were analyzed via RT-qPCR. ( E ) TonEBP WT and TonEBP KO MEFs were treated with 500 mOsm sucrose. RNA was isolated, and mRNA transcripts were analyzed via RT-qPCR. ( F ) Control and shc-Rel MEFs were treated with varying intensities of sucrose. RNA was isolated, and mRNA transcripts were analyzed via RT-qPCR. ( G ) After indicated treatments, cytoplasmic membrane fractions were isolated and analyzed via western blot. ( H ) After indicated treatments, total lysates were isolated and analyzed via western blot. ( I ) Control and shc-Rel MEFs were treated with 500 mOsm sucrose. Intracellular levels of the amino acid proline were analyzed via amino acid uptake assay. ( J ) Control and shc-Rel MEFs and TonEBP WT and KO MEFs were transfected with the Slc38a2 -promoter luciferase reporter construct, then treated with 500 mOsm sucrose. Luciferase activity was normalized to co-transfected Renilla luciferase activity. ( K ) Control and shc-Rel MEFs and TonEBP WT and KO MEFs were transfected with the Ppp1r15a -promoter luciferase reporter construct, then treated with 500 mOsm sucrose. Luciferase activity was normalized to co-transfected Renilla luciferase activity. Graph values for caspase-3 activity assays and RT-qPCR experiments in Figure 2 .

    Journal: eLife

    Article Title: PACT-mediated PKR activation acts as a hyperosmotic stress intensity sensor weakening osmoadaptation and enhancing inflammation

    doi: 10.7554/eLife.52241

    Figure Lengend Snippet: NF-κB c-Rel is a novel factor in the adaptive response to hyperosmotic stress. ( A ) Control and shPACT MEFs were treated with 500 or 600 mOsm. Nuclear fractions were isolated, and protein levels were analyzed via western blot. Insert indicates efficiency of PACT depletion from cytoplasmic extracts. ( B ) Control and shc-Rel MEFs were treated with 500 mOsm sucrose. Lysates were assayed for caspase-3 enzymatic activity. ( C ) Control and shc-Rel MEFs were treated with 500 or 600 mOsm sucrose. Nuclear fractions were isolated and protein levels were analyzed via western blot. ( D ) Control and shc-Rel MEFs were treated with 500 or 600 mOsm sucrose. RNA was isolated, and mRNA transcripts were analyzed via RT-qPCR. ( E ) TonEBP WT and TonEBP KO MEFs were treated with 500 mOsm sucrose. RNA was isolated, and mRNA transcripts were analyzed via RT-qPCR. ( F ) Control and shc-Rel MEFs were treated with varying intensities of sucrose. RNA was isolated, and mRNA transcripts were analyzed via RT-qPCR. ( G ) After indicated treatments, cytoplasmic membrane fractions were isolated and analyzed via western blot. ( H ) After indicated treatments, total lysates were isolated and analyzed via western blot. ( I ) Control and shc-Rel MEFs were treated with 500 mOsm sucrose. Intracellular levels of the amino acid proline were analyzed via amino acid uptake assay. ( J ) Control and shc-Rel MEFs and TonEBP WT and KO MEFs were transfected with the Slc38a2 -promoter luciferase reporter construct, then treated with 500 mOsm sucrose. Luciferase activity was normalized to co-transfected Renilla luciferase activity. ( K ) Control and shc-Rel MEFs and TonEBP WT and KO MEFs were transfected with the Ppp1r15a -promoter luciferase reporter construct, then treated with 500 mOsm sucrose. Luciferase activity was normalized to co-transfected Renilla luciferase activity. Graph values for caspase-3 activity assays and RT-qPCR experiments in Figure 2 .

    Article Snippet: RT-qPCR Total RNA was prepared using TRIzol reagent (Ambion) according to manufacturer’s instructions.

    Techniques: Isolation, Western Blot, Activity Assay, Quantitative RT-PCR, Transfection, Luciferase, Construct

    Additional proinflammatory gene expression programs are also dependent on PACT. ( A ) MEFs deficient in PACT were treated with the indicated stress intensity. RNA was isolated, and mRNA transcript levels analyzed via RT-qPCR.

    Journal: eLife

    Article Title: PACT-mediated PKR activation acts as a hyperosmotic stress intensity sensor weakening osmoadaptation and enhancing inflammation

    doi: 10.7554/eLife.52241

    Figure Lengend Snippet: Additional proinflammatory gene expression programs are also dependent on PACT. ( A ) MEFs deficient in PACT were treated with the indicated stress intensity. RNA was isolated, and mRNA transcript levels analyzed via RT-qPCR.

    Article Snippet: RT-qPCR Total RNA was prepared using TRIzol reagent (Ambion) according to manufacturer’s instructions.

    Techniques: Expressing, Isolation, Quantitative RT-PCR

    Hyperosmotic stress-induced adaptive gene expression occurs independent of cell type and osmolyte used. ( A ) Control and shc-Rel MEFs were treated with 5% or 10% DSS. RNA was isolated, and mRNA transcripts analyzed via RT-qPCR. ( B ) Control and shc-Rel MEFs were treated with 5% or 10% DSS. Cytoplasmic membrane fractions were isolated, and protein levels analyzed via western blot. ( C ) Control and shc-Rel MEFs were treated with 5% or 10% DSS. Total lysates were analyzed via western blot. ( D ) RAW macrophages were treated with 5% or 10% DSS for the indicated durations. RNA was isolated, and transcripts were analyzed via qPCR. ( E ) Primary tracheal epithelial cells cultured from human patients were treated with the indicated concentration of NaCl. RNA was isolated, and transcripts were analyzed via qPCR. Graph values for RT-qPCR experiments in Figure 3 .

    Journal: eLife

    Article Title: PACT-mediated PKR activation acts as a hyperosmotic stress intensity sensor weakening osmoadaptation and enhancing inflammation

    doi: 10.7554/eLife.52241

    Figure Lengend Snippet: Hyperosmotic stress-induced adaptive gene expression occurs independent of cell type and osmolyte used. ( A ) Control and shc-Rel MEFs were treated with 5% or 10% DSS. RNA was isolated, and mRNA transcripts analyzed via RT-qPCR. ( B ) Control and shc-Rel MEFs were treated with 5% or 10% DSS. Cytoplasmic membrane fractions were isolated, and protein levels analyzed via western blot. ( C ) Control and shc-Rel MEFs were treated with 5% or 10% DSS. Total lysates were analyzed via western blot. ( D ) RAW macrophages were treated with 5% or 10% DSS for the indicated durations. RNA was isolated, and transcripts were analyzed via qPCR. ( E ) Primary tracheal epithelial cells cultured from human patients were treated with the indicated concentration of NaCl. RNA was isolated, and transcripts were analyzed via qPCR. Graph values for RT-qPCR experiments in Figure 3 .

    Article Snippet: RT-qPCR Total RNA was prepared using TRIzol reagent (Ambion) according to manufacturer’s instructions.

    Techniques: Expressing, Isolation, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, Cell Culture, Concentration Assay

    MiR-1 inhibited angiogenesis-related factors at both the messenger RNA level and the protein level. a Quantitative reverse transcription PCR assay. P values were determined by an unpaired two-sided t test. b Western blotting. c Relative protein levels of vascular endothelial growth factor A ( VEGF-A ) in supernatant of gastric cancer cells transitorily transfected with pri-miR-1 plasmid determined by ELISA. Cont. control, WT wild type, * P

    Journal: Gastric Cancer

    Article Title: MicroRNA-1 acts as a tumor suppressor microRNA by inhibiting angiogenesis-related growth factors in human gastric cancer

    doi: 10.1007/s10120-017-0721-x

    Figure Lengend Snippet: MiR-1 inhibited angiogenesis-related factors at both the messenger RNA level and the protein level. a Quantitative reverse transcription PCR assay. P values were determined by an unpaired two-sided t test. b Western blotting. c Relative protein levels of vascular endothelial growth factor A ( VEGF-A ) in supernatant of gastric cancer cells transitorily transfected with pri-miR-1 plasmid determined by ELISA. Cont. control, WT wild type, * P

    Article Snippet: RNA extraction, reverse transcription, and quantitative real-time PCR Total RNA was extracted from tissues or cultured cells with Trizol reagent.

    Techniques: Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    Glutamine‐restriction improves the metabolic status in CD 8 + T cells. OVA ‐specific OT ‐1 CD 8 + T cells were cultured as shown in (Figure 1 A). A, The O 2 consumption rate ( OCR ) was measured in real time under basal conditions and in response to indicated mitochondrial inhibitors. The spare respiratory capacity ( SRC ) is shown (left panel). Data are representative of at least 2 independent experiments. B, The gene expression of mitochondrial transcription factors ( Tfam1 and Tfb2 m ) and RNA polymerase ( Polrmt ) in CD 8 + T cells cultured under glutamine‐restricted conditions. The expression of mRNA was examined by quantitative RT ‐ PCR and compared between control (Ctrl)‐cultured and dG ln‐cultured cells (mean ± SD , n = 4 per group). C, Representative flow‐cytometric profiles of mitochondrial reactive oxygen species (ROS) . Data are representative of 2 independent experiments. D, The extracellular acidification rate ( ECAR ) was measured under basal conditions and in response to 10 mmol/L glucose, 100 mmol/L glucose and 10 μmol/L oligomycin. Data are representative of 2 independent experiments. E, The gene expression of glycolysis‐related enzymes in CD 8 + T cells cultured under glutamine‐restricted conditions. The expression of mRNA was examined by quantitative RT ‐ PCR and compared between Ctrl‐ and dG ln‐cultured cells (mean ± SD , n = 4 per group). ** P

    Journal: Cancer Science

    Article Title: Reinforce the antitumor activity of CD8+ T cells via glutamine restriction, et al. Reinforce the antitumor activity of CD8+ T cells via glutamine restriction

    doi: 10.1111/cas.13827

    Figure Lengend Snippet: Glutamine‐restriction improves the metabolic status in CD 8 + T cells. OVA ‐specific OT ‐1 CD 8 + T cells were cultured as shown in (Figure 1 A). A, The O 2 consumption rate ( OCR ) was measured in real time under basal conditions and in response to indicated mitochondrial inhibitors. The spare respiratory capacity ( SRC ) is shown (left panel). Data are representative of at least 2 independent experiments. B, The gene expression of mitochondrial transcription factors ( Tfam1 and Tfb2 m ) and RNA polymerase ( Polrmt ) in CD 8 + T cells cultured under glutamine‐restricted conditions. The expression of mRNA was examined by quantitative RT ‐ PCR and compared between control (Ctrl)‐cultured and dG ln‐cultured cells (mean ± SD , n = 4 per group). C, Representative flow‐cytometric profiles of mitochondrial reactive oxygen species (ROS) . Data are representative of 2 independent experiments. D, The extracellular acidification rate ( ECAR ) was measured under basal conditions and in response to 10 mmol/L glucose, 100 mmol/L glucose and 10 μmol/L oligomycin. Data are representative of 2 independent experiments. E, The gene expression of glycolysis‐related enzymes in CD 8 + T cells cultured under glutamine‐restricted conditions. The expression of mRNA was examined by quantitative RT ‐ PCR and compared between Ctrl‐ and dG ln‐cultured cells (mean ± SD , n = 4 per group). ** P

    Article Snippet: 2.8 RNA isolation and quantitative RT‐PCR Total RNA was isolated using TRI Reagent (cat.# TR118; Molecular Research Center, Cincinnati, OH, USA) or NucleoSpin RNA XS (cat.# 740902.10; Takara Bio, Shiga, Japan) according to the manufacturer's protocols. cDNA was then synthesized using the Superscript VILO cDNA Synthesis Kit (cat.# 11755‐500; Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Flow Cytometry

    Linc-ROR shares miRNA binding sites with SOX9. a qRT-PCR analysis of the expression of linc-ROR and SOX9 in EC9706 cells transfected with 12 different miRNA mimics versus scramble control. b qRT-PCR analysis of linc-ROR and SOX9 expression after treatment with miR-145 inhibitor; a mixture of miR-15b, miR-33a, and miR-129 inhibitors (3 miR-inh mix); and inhibitor cocktail of miR-15b, miR-33a, miR-129, miR-145, and miR-206 (5 miR-inh mix). c qRT-PCR analysis of stemness-associated genes CD44, KLF4, NANOG, OCT4, and SOX2 expression following treatment with miR-145 mimics in EC9706 or 5 miR-inh mix in Eca109 cells. Transcription levels were normalized to GAPDH expression. d Prediction for five candidate miRNA-binding elements on linc-ROR transcript and SOX9 3′-UTR. e Sequence alignment of miR-15b, miR-33a, miR-129, miR-145, and miR-206 seed sequence in linc-ROR and SOX9 3′-UTR. f Amount of linc-ROR and SOX9 bound to Ago2 was determined by qRT-PCR in the presence of inhibitor cocktail of all candidate miRNAs or negative control. IgG was used as a negative control. g SOX9 protein level in EC9706 cells following treatment with linc-ROR siRNA or miR-145 mimics was measured by Western blot (top). Luciferase assay of 293 T cells co-transfected with pmiR-REPORT-SOX9 3′-UTR and indicated RNA oligos. siRNA targeting SOX9 3′-UTR was used as a positive control (bottom)

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Linc-ROR promotes esophageal squamous cell carcinoma progression through the derepression of SOX9

    doi: 10.1186/s13046-017-0658-2

    Figure Lengend Snippet: Linc-ROR shares miRNA binding sites with SOX9. a qRT-PCR analysis of the expression of linc-ROR and SOX9 in EC9706 cells transfected with 12 different miRNA mimics versus scramble control. b qRT-PCR analysis of linc-ROR and SOX9 expression after treatment with miR-145 inhibitor; a mixture of miR-15b, miR-33a, and miR-129 inhibitors (3 miR-inh mix); and inhibitor cocktail of miR-15b, miR-33a, miR-129, miR-145, and miR-206 (5 miR-inh mix). c qRT-PCR analysis of stemness-associated genes CD44, KLF4, NANOG, OCT4, and SOX2 expression following treatment with miR-145 mimics in EC9706 or 5 miR-inh mix in Eca109 cells. Transcription levels were normalized to GAPDH expression. d Prediction for five candidate miRNA-binding elements on linc-ROR transcript and SOX9 3′-UTR. e Sequence alignment of miR-15b, miR-33a, miR-129, miR-145, and miR-206 seed sequence in linc-ROR and SOX9 3′-UTR. f Amount of linc-ROR and SOX9 bound to Ago2 was determined by qRT-PCR in the presence of inhibitor cocktail of all candidate miRNAs or negative control. IgG was used as a negative control. g SOX9 protein level in EC9706 cells following treatment with linc-ROR siRNA or miR-145 mimics was measured by Western blot (top). Luciferase assay of 293 T cells co-transfected with pmiR-REPORT-SOX9 3′-UTR and indicated RNA oligos. siRNA targeting SOX9 3′-UTR was used as a positive control (bottom)

    Article Snippet: RNA isolation and quantitative real-time PCR Total RNA was isolated from cultured cells or human samples using Total RNA Kit I (Omega Bio-tek) or miRNeasy FFPE Kit (Qiagen) according to the manufacturer’s protocols, respectively. cDNA was synthesized using reverse transcriptase, after which quantitative real-time PCR (qRT-PCR) was performed using Fast SYBR qPCR mixture (CWBIO) with specific primers on 7500 Fast Real-Time PCR System (Applied Biosystems).

    Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Transfection, Sequencing, Negative Control, Western Blot, Luciferase, Positive Control