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  • 99
    Zymo Research rna
    RocA clamps eIF4A on polypurine motif even after ATP hydrolysis (a, b) Direct measurement of the <t>eIF4A/RNA</t> affinity by fluorescence polarization for eIF4A and 5′ FAM-labeled RNAs in the presence or absence of RocA. Data represent mean and S.D. (n = 3). (c) Motif enrichments along entire 4-mer motifs in Bind-n-Seq with ADP + Pi and highest-scoring elements (inset). (d) Competition assay with unlabeled RNA. Data represent mean (n = 3). (e) Ribosome toeprinting assay performed in RRL in the presence of GMP-PNP in the presence or absence of 3 μM RocA treatment. (f) Relative <t>RNase</t> I cleavage protected by eIF4A/RocA complex on mRNA containg one AGAGAG at the middle in footprinting assay. See the original data in Extended Data Figure 9f .
    Rna, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 531 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs nebnext ultra rna library prep kit for illumina
    Optimization of SHERRY and comparison with <t>NEBNext.</t> ( A ) Gene number detected by SHERRY under various experimental conditions. Each condition consisted of three replicates of 10 ng HEK293T total <t>RNA.</t> ( B ) Comparison of sequencing indicators between SHERRY and NEBNext with 10 ng and 200 ng HEK293T total RNA input. Each condition consisted of three replicates and down-sampled to 2 million total reads.
    Nebnext Ultra Rna Library Prep Kit For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher total rna
    Star-PAP regulated <t>miR-449a/34a</t> expressions. (A) Overexpression of Star-PAP for 24 h. The expression of miR-449a/34a was detected by qPCR. (B) Decreased levels of miR-449a/34a following Star-PAP knockdown by siRNAs for 48 h. (C) Cells were co-transfected with either pFlagcmv2-Star-PAP and 80 ng plasmid carrying either WT or Mut 3′-UTR of TPD52. The relative firefly luciferase activity normalized with Renilla luciferase was measured 24 h after transfection. (D) <t>RNA</t> pull down assay was performed by 5′-biotin-miR-449a and biotin-Scramble. 293 FT cells were transfected with pFlagcmv2-Star-PAP for 24 h. Cells were lysed in RIPA buffer, then incubated with biotin-miR-449a or biotin-scramble for 4 h, before that they were pre-incubated with miR-449a or Scramble for 1 h, followed by adding avidin beads, and finally examined by western blot. (E) Cells were co-transfected with Star-PAP and miR-449a mimic or inhibitor, the TPD52 mRNA level was detected by qPCR. (F) The same treatment as with E, and the protein levels were examined. (G) Quantification of TPD52 protein level in F by NIH ImageJ software. Data are means±s.d. ( n =3) with three independent repeats, * P
    Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 496855 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore rna
    Requirement of ER stress for efficient brown adipogenesis. ( A and B ) HB2 brown preadipocytes were treated with or without 5 mM of 4-PBA or 100 μM of capsaicin or both for 8 days in the presence of insulin (20 nM). ( C and D ) HB2 brown preadipocytes were transfected with scrambled <t>RNA</t> or <t>siRNA</t> for Xbp1. Cells were treated with or without 100 μM of capsaicin in the presence of insulin (20 nM) for 8 days. ( A and C ) Oil Red O staining of cells on day 8 was performed, and the dye intensity was quantified (n = 2). ( B and D ) The expression level of Ucp1 was examined by RT-qPCR analysis. Black bar: vehicle; Hatched bar: capsaicin. The data are presented as the mean ± SE (n = 4). * P
    Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5994 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore total rna
    The three core TS cell transcription factors Cdx2, Elf5 and Tead4 are insufficient to complete reprogramming to induce a stable TS cell phenotype in bulk culture. ( a ) iCdx2 cells, shown previously to be capable of placental contribution 18 , and iCdx2/iRaf cells were transfected with an Elf5-2A-Tead4-2A-Hygro R (E+T) or Hygro R -only control (vec) expression construct, subjected to the trans-differentiation protocol for 6 days on antibiotic-resistant MEFs, <t>FACS-purified</t> from MEFs and assessed for gatekeeper gene expression levels by RT–qPCR. Details of overexpressed genes resulting from the various conditions are given in red. Elf5 and Tead4 expression, in conjunction with Cdx2 activation, improves the activation of some of the remaining gatekeeper genes. Values are of three biological replicates and expressed as percent of TS cell expression levels combined of the two independent TS cell lines (mean±s.e.m.). ( b ) Activation of TS cell transcription factors in the same cells as in ( a ). Hyperactivation of Tcfap2c and Hand1 (in a ) indicate onset of terminal trophoblast differentiation. Values expressed as in a . ( c ) Dynamics of methylation reprogramming of these cells at the ES-DMRs identified by meDIP-seq and assessed by Sequenom analysis as in Fig. 4c . ( d ) Assessment of phenotypic stability of reprogrammed TSL (iCdx2) cells. Upon removal of the MEF layer, the tight epithelial colony shape that is characteristic of bona fide TS cells was rapidly lost, and cells failed to maintain the co-expression of Cdx2 and Elf5 that is required to preserve the proliferative, self-renewing TS cell state, and instead started to differentiate. Scale bars: 200 μm (phase contrast), 100 μm (immunofluorescence). ( e ) <t>RNA-seq</t> analysis of iCdx2 (5ECER4G20) and iCdx2/iRaf ES cells grown for 5 days on MEFs, and then replated for 3 days on gelatinized TC plastic. Note the instability of gene expression, and specifically the downregulation of critical TS cell genes such as Eomes , Elf5 , Esrrb and Sox2 upon transfer of reprogrammed TSL cells from MEFs onto TC plastic.
    Total Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 27839 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Agilent technologies rna integrity
    Relationship between SDV and <t>RIN</t> . Comparison of the SDV and RIN integrity metrics for a panel of seven <t>RNA</t> samples.
    Rna Integrity, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 95/100, based on 43937 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen qiaamp viral rna mini kit
    Amplification of a serial dilution of the EUROHEP standard with HBV-specific primers in a nested PCR following extraction with the Chemagic <t>DNA/RNA</t> kit and the <t>QIAamp</t> DNA Blood Mini kit. (A) Chemagen extracts. (B) QIAGEN extracts. Lane M shows a 100-bp ladder, and lane C shows results for the negative control. Lanes 1 through 10 show results for different viral loads expressed in numbers of copies per ml: 1, 4 × 10 5 ; 2, 4 × 10 4 ; 3, 4 × 10 3 ; 4, 4 × 10 2 ; 5, 2 × 10 2 ; 6, 1 × 10 2 ; 7, 8 × 10 1 ; 8, 4 × 10 1 ; 9, 2 × 10 1 ; 10, 1 × 10 1 .
    Qiaamp Viral Rna Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 23436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher purelink rna mini kit
    IL-10 mRNA expression by B10 cells after LPS and/or CD40L stimulation. Cultured mouse slpenocytes were treated with E. coli LPS (10 µg/ml) and/or CD40L (1 µg/ml) for 2 days and CD19 + CD1d hi CD5 + cells in the cultured splenocytes were sorted out using flow cytometry. Total <t>RNA</t> were isolated from sorted cells using a <t>Purelink</t> RNA mini kit (Life Technology) and IL-10 expression was determined by real time PCR. At least 100,000 cells were sorted from each sample for PCR. Data are presented as mean ± SEM, n = 6. Student t-test, *P
    Purelink Rna Mini Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16743 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher high capacity rna to cdna kit
    Sequence alignment of HIV vmiRs with consensus genomic sequence from HIV-1 subtypes and absolute quantitation of miRNAs by Real Time RT-PCR. A : Alignment of vmiR sequences of GU-rich tract is consistent with consensus genomic sequence from HIV-1 subtypes A-J, 533 isolates. B : VmiR99 is 90–100% identical to 96% of HIV-1 sequences. Sequences within the GU tract, vmiR88, vmiR99 and ssRNA40 were aligned with 196, 201, 254 and 272 genome sequences, respectively. C : Genomic <t>RNA</t> of HIV-1 BaL strain was scanned for every 21-bp RNA segment and the distribution of base compositions (46.5±11.8% G+U) is shown. VmiR-TAR is GU-poor (35%). VmiR88 and vmiR99 are GU-rich (71% and 76% G+U, respectively). Absolute quantitation of miRNAs was determined by Real Time RT-PCR. After first strand <t>cDNA</t> synthesis, amplification ( D, F, H ) and absolute quantitation ( E, G, I ) of vmiR-TAR ( D–E ) vmiR88 ( F–G ), vmiR99 ( H–I ) and RNA40 (I) was standardized using synthetic miR oligonucleotides in the miRCURY LNA Universal RT microRNA PCR method (Exiqon) on an ABI 7900HT FAST Real Time PCR system. ΔR n is the change in normalized reporter fluorescence intensity. C T is the threshold cycle in which the amplification curve crosses the dashed horizontal line. Data depict a representative experiment done in duplicate.
    High Capacity Rna To Cdna Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18369 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies rna 6000 nano kit
    RNA quality of cultivated tissue slices. RNA quality was determined by a Bioanalyzer 2100 using the <t>RNA</t> 6000 <t>Nano-Kit</t> (Agilent Technologies) and revealed good quality before the DNase digestion was performed ( a ). After the DNase digestion, the RNA quality was strongly reduced ( b ). The left graphs show untreated peritumoral brain tissue, the right graphs the corresponding GBM tissue.
    Rna 6000 Nano Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 13207 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Illumina Inc rna seq libraries
    Gene body H3K36me3 level is negatively correlated with age-dependent <t>mRNA</t> expression change in Drosophila heads. ( A ) All genes with mappable H3K36me3 ChIP–chip and <t>RNA-seq</t> reads are plotted in the order of normalized H3K36me3 levels at the young
    Rna Seq Libraries, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 95/100, based on 14521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Promega sv total rna isolation system
    <t>VFP4-controlled</t> genes ATL31 and At2g32030 . Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of ATL31 and At2g32030 expression in roots of A , wild-type Col-0 and vfp4-1 plants and B , wild-type Col-0 and VFP4 OE-6 plants. C , RT-qPCR analysis of ATL31 and At2g32030 expression in roots of the wild-type Col-0, ATL31 OE-4, and ATL31 OE-5 and Col-0 and At2g32030 OE-9 plants, respectively. The levels of expression were normalized to the internal reference genes ACT7 and 18S <t>RNA</t> . The expression level of each tested gene in the wild-type Col-0 is set to 1.0, and error bars represent standard error of the mean of independent biological replicates, n = 3.
    Sv Total Rna Isolation System, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 13726 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t7 rna polymerase
    Dominant-negative effect of L-protein mutants. (A) Inactive mutants were transfected in a ratio of 1:1 with wild-type L protein. As a control, a PCR fragment of the L gene lacking the upstream <t>T7</t> RNA polymerase promoter was transfected 1:1 with the complete
    T7 Rna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Illumina Inc sequencing rna
    Validation of gene expression changes estimated with CANEapp with quantitative real-time PCR. a <t>RNA-seq</t> analysis of hippocampi of Alzheimer’s disease patients and controls. Hippocampal tissue from 4 AD patients and 4 control individuals was used to extract total RNA and perform ribodepletion and strand-specific library preparation. Single-end RNA sequencing was performed on <t>Illumina</t> HiSeq 2000. Fold changes of expression for 2 downregulated and 4 upregulated genes measured with real-time PCR was compared with expression values generated by CANEapp. b RNA-seq of developing mouse cortex. Tissue from 4 embryonic day 17 and 3 adult mouse cortical samples was processed to extract polyA-selected RNA and generate paired-end unidirectional sequencing data with Illumina Genome Analyzer IIx. Gene expression estimates of 4 downregulated and 4 upregulated genes were compared between CANEapp and real-time PCR. c Fold changes of gene expression for RNA-seq of liver of rats treated with two DNA-damage compounds. The data was produced by paired-end sequencing of polyA-selected RNA on Illumina HiSeq 2000. Fold changes of expression for 2 downregulated and 4 upregulated genes were compared between CANEapp and real-time PCR. R 2 -coefficient of determination
    Sequencing Rna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rna  (Qiagen)
    99
    Qiagen rna
    NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by <t>RNA-seq</t> analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated <t>hiPSC-RPE.</t> Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p
    Rna, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 49618 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Zymo Research direct zol rna miniprep kit
    HCMV infection leads to an upregulation of polycomb group proteins. (A and B) HFF cells were infected with the laboratory HCMV strain AD169 (A) and the clinical strain TB40/E (B) at an MOI of 3. Samples were harvested at the indicated time points postinfection, and whole-cell extracts were subjected to subsequent SDS-PAGE and Western blot analysis. Cellular PcG proteins were detected by the indicated antibodies. The detection of viral immediate early (IE1), early (pUL44), and late (pp28, MCP) proteins was used to monitor progression of the replication cycle. β-Actin served as a loading control. (C) HFF cells were infected with AD169 at an MOI of 3 and harvested at the indicated time points postinfection. <t>RNA</t> was isolated using TRIzol and the <t>Direct-zol</t> RNA <t>miniprep</t> kit (Zymo Research) and synthesized into cDNA by RT-PCR. Transcript levels were assessed by SYBR green PCR, and relative mRNA levels were calculated by normalization against the value for the housekeeping gene GAPDH. Values are derived from biological triplicates and represent mean values ± standard deviations (SD). Statistical analysis was performed by ordinary one-way analysis of variance (ANOVA). n.s., not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (D) HFF cells were infected with AD169 at an MOI of 3 and treated with cycloheximide (CHX) (150 μg/ml) in parallel. After the indicated time points postinfection, whole-cell lysates were prepared and subjected to Western blot analysis. Cellular and viral proteins were detected by the indicated antibodies. IE proteins (IE1 or IE2) served as controls for blocked de novo protein synthesis, while pp65 ensured the income of tegument proteins. β-Actin served as a loading control. (E) HFF cells were infected with either AD169 or UV-inactivated AD169 at an MOI of 1. Whole-cell extracts were generated at the indicated time points and subjected to Western blot analysis. Detection of proteins was equivalent to that for panel D.
    Direct Zol Rna Miniprep Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 7194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen allprep dna rna mini kit
    A scatterplot showing the ratio of live, potentially active bacteria <t>(RNA/DNA)</t> in tumor vs . non-tumor tissue.
    Allprep Dna Rna Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 7493 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega t7 rna polymerase
    A codon-modified GAr sequence influences EBNA1 synthesis. ( A ) IVT assay of pcDNA3 expression constructs encoding EBNA1 (E1) (lane 1), E1ΔGA (lane 2), E1-GAr(100N) (lane 3), E1-GAr(100M) (lane 4), E1-GAr(200N) (lane 5), E1-GAr(200M) (lane 6), E1-GAr(300N) (lane 7), E1-GAr(300M) (lane 8), E1-GAr(400N) (lane 9), E1-GAr(400M) (lane 10), E1-GAr(500N) (lane 11), or E1-GAr(500M) (lane 12). The constructs were transcribed and translated in vitro with <t>T7</t> RNA polymerase by using a coupled transcription/translation reticulocyte lysate system. 35 S-methionine-labeled proteins were visualized by autoradiography. ( B and C ) Band intensities from the IVT assay were quantified by densitometric analysis using Imagequant software (Molecular Dynamics) and graphed to demonstrate absolute intensities ( B ) or relative fold increase of EBNA1 encoded by codon-modified GAr domains compared with EBNA1 encoded by native GAr domains ( C ). ( D ) Western blot of EBV-negative HEK293 cells transfected with expression constructs encoding E1-GFP (lane 1), E1ΔGA-GFP (lane 2), E1-GAr(100N)-GFP (lane 3), E1-GAr(100M)-GFP (lane 4), E1-GAr(200N)-GFP (lane 5), E1-GAr(200M)-GFP (lane 6), E1-GAr(300N)-GFP (lane 7), E1-GAr(300M)-GFP (lane 8), E1-GAr(400N)-GFP (lane 9), E1-GAr(400M)-GFP (lane 10), E1-GAr(500N)-GFP (lane 11), or E1-GAr(500M)-GFP (lane 12) with a GFP antibody ( Upper ) or a monoclonal actin antibody ( Lower ). Molecular weight markers M r (kDa) are indicated on the left. ( E ) Band intensities after immunoblotting were quantified as described for B . Representative data from one of four experiments are presented here.
    T7 Rna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 10131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Zymo Research total rna
    YAP1 is activated by platelets and is indispensable for platelet-induced anoikis resistance. a , b Western blot analysis of phosphorylated YAP1 (S127 and S397) and total YAP1 in HEYA8 a and OVCAR8 b cells after two hours under low-attachment conditions with or without platelet co-incubation. GAPDH was used as a loading control ( n = 5). c , d Western blot analysis of phosphorylated YAP1 (S127) and total YAP1 in SKOV3, MDAH-2774, OVCAR4, SW620 c and OVCAR5, OVCA432 and RKO d GAPDH was used as a loading control ( n = 3). e , f Immunofluorescence staining of YAP1 in HEYA8 e and OVCAR8 f cells after two hours under low-attachment conditions with ( lower panels ) or without ( upper panels ) platelet co-incubation. Inlets showing higher magnification of cells on the right side of the panels. Nuclear counterstain was done using Hoechst 33342 ( n = 3). Scale bars = 20 µm. g <t>QRT–PCR</t> and western blot analysis in HEYA8 cells showing efficiency of YAP1 knockdown on the <t>RNA</t> and protein level using two different siRNAs ( n = 3). h Bar graphs showing number of dead (SYTOX Red positive, black ) and living (SYTOX Red negative, red ) HEYA8 cells after 72 h of low attachment and 96 h after siRNA transfection ( n = 3, two-sided Student’s t -test). i QRT–PCR and western blot analysis in OVCAR8 cells showing efficiency of YAP1 knockdown on the RNA and protein level using two different siRNAs ( n = 3). j Bar graphs showing number of dead (SYTOX Red positive, black ) and living (SYTOX Red negative, red ) OVCAR8 cells after 72 h of low attachment and 96 h after siRNA transfection ( n = 3, two-sided Student’s t -test). Bars and error bars represent mean values and the corresponding SEMs (* p
    Total Rna, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 7667 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore rna extraction
    YAP1 is activated by platelets and is indispensable for platelet-induced anoikis resistance. a , b Western blot analysis of phosphorylated YAP1 (S127 and S397) and total YAP1 in HEYA8 a and OVCAR8 b cells after two hours under low-attachment conditions with or without platelet co-incubation. GAPDH was used as a loading control ( n = 5). c , d Western blot analysis of phosphorylated YAP1 (S127) and total YAP1 in SKOV3, MDAH-2774, OVCAR4, SW620 c and OVCAR5, OVCA432 and RKO d GAPDH was used as a loading control ( n = 3). e , f Immunofluorescence staining of YAP1 in HEYA8 e and OVCAR8 f cells after two hours under low-attachment conditions with ( lower panels ) or without ( upper panels ) platelet co-incubation. Inlets showing higher magnification of cells on the right side of the panels. Nuclear counterstain was done using Hoechst 33342 ( n = 3). Scale bars = 20 µm. g <t>QRT–PCR</t> and western blot analysis in HEYA8 cells showing efficiency of YAP1 knockdown on the <t>RNA</t> and protein level using two different siRNAs ( n = 3). h Bar graphs showing number of dead (SYTOX Red positive, black ) and living (SYTOX Red negative, red ) HEYA8 cells after 72 h of low attachment and 96 h after siRNA transfection ( n = 3, two-sided Student’s t -test). i QRT–PCR and western blot analysis in OVCAR8 cells showing efficiency of YAP1 knockdown on the RNA and protein level using two different siRNAs ( n = 3). j Bar graphs showing number of dead (SYTOX Red positive, black ) and living (SYTOX Red negative, red ) OVCAR8 cells after 72 h of low attachment and 96 h after siRNA transfection ( n = 3, two-sided Student’s t -test). Bars and error bars represent mean values and the corresponding SEMs (* p
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    YAP1 is activated by platelets and is indispensable for platelet-induced anoikis resistance. a , b Western blot analysis of phosphorylated YAP1 (S127 and S397) and total YAP1 in HEYA8 a and OVCAR8 b cells after two hours under low-attachment conditions with or without platelet co-incubation. GAPDH was used as a loading control ( n = 5). c , d Western blot analysis of phosphorylated YAP1 (S127) and total YAP1 in SKOV3, MDAH-2774, OVCAR4, SW620 c and OVCAR5, OVCA432 and RKO d GAPDH was used as a loading control ( n = 3). e , f Immunofluorescence staining of YAP1 in HEYA8 e and OVCAR8 f cells after two hours under low-attachment conditions with ( lower panels ) or without ( upper panels ) platelet co-incubation. Inlets showing higher magnification of cells on the right side of the panels. Nuclear counterstain was done using Hoechst 33342 ( n = 3). Scale bars = 20 µm. g <t>QRT–PCR</t> and western blot analysis in HEYA8 cells showing efficiency of YAP1 knockdown on the <t>RNA</t> and protein level using two different siRNAs ( n = 3). h Bar graphs showing number of dead (SYTOX Red positive, black ) and living (SYTOX Red negative, red ) HEYA8 cells after 72 h of low attachment and 96 h after siRNA transfection ( n = 3, two-sided Student’s t -test). i QRT–PCR and western blot analysis in OVCAR8 cells showing efficiency of YAP1 knockdown on the RNA and protein level using two different siRNAs ( n = 3). j Bar graphs showing number of dead (SYTOX Red positive, black ) and living (SYTOX Red negative, red ) OVCAR8 cells after 72 h of low attachment and 96 h after siRNA transfection ( n = 3, two-sided Student’s t -test). Bars and error bars represent mean values and the corresponding SEMs (* p
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    Image Search Results


    RocA clamps eIF4A on polypurine motif even after ATP hydrolysis (a, b) Direct measurement of the eIF4A/RNA affinity by fluorescence polarization for eIF4A and 5′ FAM-labeled RNAs in the presence or absence of RocA. Data represent mean and S.D. (n = 3). (c) Motif enrichments along entire 4-mer motifs in Bind-n-Seq with ADP + Pi and highest-scoring elements (inset). (d) Competition assay with unlabeled RNA. Data represent mean (n = 3). (e) Ribosome toeprinting assay performed in RRL in the presence of GMP-PNP in the presence or absence of 3 μM RocA treatment. (f) Relative RNase I cleavage protected by eIF4A/RocA complex on mRNA containg one AGAGAG at the middle in footprinting assay. See the original data in Extended Data Figure 9f .

    Journal: Nature

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    doi: 10.1038/nature17978

    Figure Lengend Snippet: RocA clamps eIF4A on polypurine motif even after ATP hydrolysis (a, b) Direct measurement of the eIF4A/RNA affinity by fluorescence polarization for eIF4A and 5′ FAM-labeled RNAs in the presence or absence of RocA. Data represent mean and S.D. (n = 3). (c) Motif enrichments along entire 4-mer motifs in Bind-n-Seq with ADP + Pi and highest-scoring elements (inset). (d) Competition assay with unlabeled RNA. Data represent mean (n = 3). (e) Ribosome toeprinting assay performed in RRL in the presence of GMP-PNP in the presence or absence of 3 μM RocA treatment. (f) Relative RNase I cleavage protected by eIF4A/RocA complex on mRNA containg one AGAGAG at the middle in footprinting assay. See the original data in Extended Data Figure 9f .

    Article Snippet: The reaction was treated with 1 μl of 0.001 U/μl RNase I (Epicentere) at room temperature for 5 min. After quenching the digestion by the addition of 1 μl of SUPERase In RNase Inhibitor (Invitrogen), RNA was extracted by Oligo clean & concentrator (Zymo Research) and reverse transcribed by ProtoScript II (NEB) with 5′ 6-FAM labeled primer (5′-6-FAM-ATGCAGAAAAATCACGGC-3′) according to manufacturer’s intruction.

    Techniques: Fluorescence, Labeling, Competitive Binding Assay, Toeprinting Assay, Footprinting

    Characterization of toeprinting assay (a) Diagram of the reporters used in this study. (b, and c) In vitro translation in RRL with mRNAs containing seven polypurine motif (AGAGAG) insertions (b) and qPCR from the samples (c). (d) Dideoxy terminated sequencing of RNA by reverse transcription verified the toeprinting product length terminated by 48S ribosomes. (e) Ribosome toeprinting assay performed in RRL in the presence of m7-GTP in the presence or absence of 3 μM RocA treatment. (f) Toeprinting assay using 10 μM recombinant eIF4A in the presence or absence of 10 μM RocA treatment. (g) Toeprinting assay (top) and RNase I footprinting assay (bottom) using 10 μM recombinant eIF4A with mRNA containing one AGAGAG motif at the middle in the presence or absence of 10 μM RocA treatment. (h and i) Toeprinting assay using 10 μM recombinant eIF4A (VX 4 GKT) or (D296A-T298K) with mRNA containing seven AGAGAG motifs in the presence or absence of 10 μM RocA treatment. (j) Pre-formation of the complex with RocA and eIF4A (VX 4 GKT) or (D296A-T298K) on the mRNA bearing seven polypurine motifs represses the translation from the mRNA in RRL. (k) Basal translation level from mRNA containing seven AGAGAG with the supplementation of recombinant eIF4A. (l) In vitro translation in RRL with mRNAs with single polypurine motif (AGAGAG) insertion at the different positions in 5′ UTR (m) Basal translation level from mRNAs bearing PV IRES and PV IRES with three AGAGAG. In b-c and h-j, data represent mean and S.D. (n = 3).

    Journal: Nature

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    doi: 10.1038/nature17978

    Figure Lengend Snippet: Characterization of toeprinting assay (a) Diagram of the reporters used in this study. (b, and c) In vitro translation in RRL with mRNAs containing seven polypurine motif (AGAGAG) insertions (b) and qPCR from the samples (c). (d) Dideoxy terminated sequencing of RNA by reverse transcription verified the toeprinting product length terminated by 48S ribosomes. (e) Ribosome toeprinting assay performed in RRL in the presence of m7-GTP in the presence or absence of 3 μM RocA treatment. (f) Toeprinting assay using 10 μM recombinant eIF4A in the presence or absence of 10 μM RocA treatment. (g) Toeprinting assay (top) and RNase I footprinting assay (bottom) using 10 μM recombinant eIF4A with mRNA containing one AGAGAG motif at the middle in the presence or absence of 10 μM RocA treatment. (h and i) Toeprinting assay using 10 μM recombinant eIF4A (VX 4 GKT) or (D296A-T298K) with mRNA containing seven AGAGAG motifs in the presence or absence of 10 μM RocA treatment. (j) Pre-formation of the complex with RocA and eIF4A (VX 4 GKT) or (D296A-T298K) on the mRNA bearing seven polypurine motifs represses the translation from the mRNA in RRL. (k) Basal translation level from mRNA containing seven AGAGAG with the supplementation of recombinant eIF4A. (l) In vitro translation in RRL with mRNAs with single polypurine motif (AGAGAG) insertion at the different positions in 5′ UTR (m) Basal translation level from mRNAs bearing PV IRES and PV IRES with three AGAGAG. In b-c and h-j, data represent mean and S.D. (n = 3).

    Article Snippet: The reaction was treated with 1 μl of 0.001 U/μl RNase I (Epicentere) at room temperature for 5 min. After quenching the digestion by the addition of 1 μl of SUPERase In RNase Inhibitor (Invitrogen), RNA was extracted by Oligo clean & concentrator (Zymo Research) and reverse transcribed by ProtoScript II (NEB) with 5′ 6-FAM labeled primer (5′-6-FAM-ATGCAGAAAAATCACGGC-3′) according to manufacturer’s intruction.

    Techniques: Toeprinting Assay, In Vitro, Real-time Polymerase Chain Reaction, Sequencing, Recombinant, Footprinting

    Optimization of SHERRY and comparison with NEBNext. ( A ) Gene number detected by SHERRY under various experimental conditions. Each condition consisted of three replicates of 10 ng HEK293T total RNA. ( B ) Comparison of sequencing indicators between SHERRY and NEBNext with 10 ng and 200 ng HEK293T total RNA input. Each condition consisted of three replicates and down-sampled to 2 million total reads.

    Journal: bioRxiv

    Article Title: RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids

    doi: 10.1101/843474

    Figure Lengend Snippet: Optimization of SHERRY and comparison with NEBNext. ( A ) Gene number detected by SHERRY under various experimental conditions. Each condition consisted of three replicates of 10 ng HEK293T total RNA. ( B ) Comparison of sequencing indicators between SHERRY and NEBNext with 10 ng and 200 ng HEK293T total RNA input. Each condition consisted of three replicates and down-sampled to 2 million total reads.

    Article Snippet: Then we plotted a heatmap of the distance matrix ( ) between different cell types and library preparation methods.

    Techniques: Sequencing

    Coverage evenness optimization of SHERRY (10 ng HEK293T total RNA input). ( A ) Normalized transcript coverage of standard SHERRY, SHERRY using TSO-RT method and NEBNext kit. ( B ) The coverage of GAPDH transcript calculated from SHERRY and TSO-RT SHERRY. ( C ) Comparison of sequencing indicators between SHERRY (n=3) and TSO-RT SHERRY (n=2).

    Journal: bioRxiv

    Article Title: RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids

    doi: 10.1101/843474

    Figure Lengend Snippet: Coverage evenness optimization of SHERRY (10 ng HEK293T total RNA input). ( A ) Normalized transcript coverage of standard SHERRY, SHERRY using TSO-RT method and NEBNext kit. ( B ) The coverage of GAPDH transcript calculated from SHERRY and TSO-RT SHERRY. ( C ) Comparison of sequencing indicators between SHERRY (n=3) and TSO-RT SHERRY (n=2).

    Article Snippet: Then we plotted a heatmap of the distance matrix ( ) between different cell types and library preparation methods.

    Techniques: Sequencing

    Functional comparison between SHERRY and NEBNext. ( A ) Correlation of normalized gene counts among duplicates of SHERRY, which start from 200 ng HEK293T total R NA input. ( B ) Correlation of normalized genes counts (average of three replicates) between SHERRY and NEBNext within the two cell types. The input was 200 ng total RNA. ( C ) Differentially expressed genes of HeLa and HEK293T detected by SHERRY and NEBNext kit (200 ng input) are plotted into Venn Diagram. Colored area represents genes identified by both methods. Gene numbers are listed on corresponding part. ( D ) Heatmap of differentially expressed genes detected by SHERRY while missed by NEBNext kit. The Color bar indicates Z-score. ( E ) Heatmap of differentially expressed genes detected by NEBNext kit while missed by SHERRY. The Color bar indicates Z-score.

    Journal: bioRxiv

    Article Title: RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids

    doi: 10.1101/843474

    Figure Lengend Snippet: Functional comparison between SHERRY and NEBNext. ( A ) Correlation of normalized gene counts among duplicates of SHERRY, which start from 200 ng HEK293T total R NA input. ( B ) Correlation of normalized genes counts (average of three replicates) between SHERRY and NEBNext within the two cell types. The input was 200 ng total RNA. ( C ) Differentially expressed genes of HeLa and HEK293T detected by SHERRY and NEBNext kit (200 ng input) are plotted into Venn Diagram. Colored area represents genes identified by both methods. Gene numbers are listed on corresponding part. ( D ) Heatmap of differentially expressed genes detected by SHERRY while missed by NEBNext kit. The Color bar indicates Z-score. ( E ) Heatmap of differentially expressed genes detected by NEBNext kit while missed by SHERRY. The Color bar indicates Z-score.

    Article Snippet: Then we plotted a heatmap of the distance matrix ( ) between different cell types and library preparation methods.

    Techniques: Functional Assay

    Performance of SHERRY with large RNA input. ( A ) Coefficient of variation (CV) across three replicates was plotted against the mean value of each gene’s FPKM (Fragments Per Kilobase of transcript per Million mapped reads). All experiments used HEK293T total RNA as input. ( B ) Genes detected by SHERRY in three replicates of 200 ng HEK293T or HeLa total RNA are plotted in Venn Diagrams. Numbers of common genes are indicated. ( C ) Common genes detected by SHERRY and NEBNext in the three replicates of 200 ng HEK293T or HeLa total RNA. ( D ) Distance heatmap of samples prepared by SHERRY or NEBNext for three replicates using 200 ng HEK293T or HeLa total RNA. The color bar indicates the Euclidian distance. ( E ) Correlation of gene expression fold-change identified by SHERRY and NEBNext. Involved genes are differentially expressed genes between HEK293T and HeLa detected by both methods.

    Journal: bioRxiv

    Article Title: RNA Sequencing by Direct Tagmentation of RNA/DNA Hybrids

    doi: 10.1101/843474

    Figure Lengend Snippet: Performance of SHERRY with large RNA input. ( A ) Coefficient of variation (CV) across three replicates was plotted against the mean value of each gene’s FPKM (Fragments Per Kilobase of transcript per Million mapped reads). All experiments used HEK293T total RNA as input. ( B ) Genes detected by SHERRY in three replicates of 200 ng HEK293T or HeLa total RNA are plotted in Venn Diagrams. Numbers of common genes are indicated. ( C ) Common genes detected by SHERRY and NEBNext in the three replicates of 200 ng HEK293T or HeLa total RNA. ( D ) Distance heatmap of samples prepared by SHERRY or NEBNext for three replicates using 200 ng HEK293T or HeLa total RNA. The color bar indicates the Euclidian distance. ( E ) Correlation of gene expression fold-change identified by SHERRY and NEBNext. Involved genes are differentially expressed genes between HEK293T and HeLa detected by both methods.

    Article Snippet: Then we plotted a heatmap of the distance matrix ( ) between different cell types and library preparation methods.

    Techniques: Expressing

    Hierarchical cluster of representative mRNA and miRNA expression across biological replicate samples. ( A ) Heatmap of representative mRNAs. ( B ) Heatmap of representative miRNAs. RNA expression level is represented by colors, with bright blue indicating high values and bright yellow indicating low values.

    Journal: Scientific Reports

    Article Title: Integrated analysis of hepatic mRNA and miRNA profiles identified molecular networks and potential biomarkers of NAFLD

    doi: 10.1038/s41598-018-25743-8

    Figure Lengend Snippet: Hierarchical cluster of representative mRNA and miRNA expression across biological replicate samples. ( A ) Heatmap of representative mRNAs. ( B ) Heatmap of representative miRNAs. RNA expression level is represented by colors, with bright blue indicating high values and bright yellow indicating low values.

    Article Snippet: Libraries for mRNA and miRNA sequencing were respectively generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB,USA) and the NEBNext Multiplex Small RNA Library Prep Kit for Illumina (NEB,USA) according to the manufacturer’s instructions.

    Techniques: Expressing, RNA Expression

    Star-PAP regulated miR-449a/34a expressions. (A) Overexpression of Star-PAP for 24 h. The expression of miR-449a/34a was detected by qPCR. (B) Decreased levels of miR-449a/34a following Star-PAP knockdown by siRNAs for 48 h. (C) Cells were co-transfected with either pFlagcmv2-Star-PAP and 80 ng plasmid carrying either WT or Mut 3′-UTR of TPD52. The relative firefly luciferase activity normalized with Renilla luciferase was measured 24 h after transfection. (D) RNA pull down assay was performed by 5′-biotin-miR-449a and biotin-Scramble. 293 FT cells were transfected with pFlagcmv2-Star-PAP for 24 h. Cells were lysed in RIPA buffer, then incubated with biotin-miR-449a or biotin-scramble for 4 h, before that they were pre-incubated with miR-449a or Scramble for 1 h, followed by adding avidin beads, and finally examined by western blot. (E) Cells were co-transfected with Star-PAP and miR-449a mimic or inhibitor, the TPD52 mRNA level was detected by qPCR. (F) The same treatment as with E, and the protein levels were examined. (G) Quantification of TPD52 protein level in F by NIH ImageJ software. Data are means±s.d. ( n =3) with three independent repeats, * P

    Journal: Biology Open

    Article Title: Star-PAP regulates tumor protein D52 through modulating miR-449a/34a in breast cancer

    doi: 10.1242/bio.045914

    Figure Lengend Snippet: Star-PAP regulated miR-449a/34a expressions. (A) Overexpression of Star-PAP for 24 h. The expression of miR-449a/34a was detected by qPCR. (B) Decreased levels of miR-449a/34a following Star-PAP knockdown by siRNAs for 48 h. (C) Cells were co-transfected with either pFlagcmv2-Star-PAP and 80 ng plasmid carrying either WT or Mut 3′-UTR of TPD52. The relative firefly luciferase activity normalized with Renilla luciferase was measured 24 h after transfection. (D) RNA pull down assay was performed by 5′-biotin-miR-449a and biotin-Scramble. 293 FT cells were transfected with pFlagcmv2-Star-PAP for 24 h. Cells were lysed in RIPA buffer, then incubated with biotin-miR-449a or biotin-scramble for 4 h, before that they were pre-incubated with miR-449a or Scramble for 1 h, followed by adding avidin beads, and finally examined by western blot. (E) Cells were co-transfected with Star-PAP and miR-449a mimic or inhibitor, the TPD52 mRNA level was detected by qPCR. (F) The same treatment as with E, and the protein levels were examined. (G) Quantification of TPD52 protein level in F by NIH ImageJ software. Data are means±s.d. ( n =3) with three independent repeats, * P

    Article Snippet: For detection of mature miR-449a/34a, total RNA was subjected to reverse TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems), and TaqMan MicroRNA Assay Kit (Applied Biosystems) was used for qPCR analysis.

    Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Pull Down Assay, Incubation, Avidin-Biotin Assay, Western Blot, Software

    Ang II- and EGF-induced Akt phosphorylation and its inhibition by RV occur independent of Nox4. Knock-down of Nox4 was achieved by treating cells with 50 nM small interfering RNA (siRNA) against Nox4 or 50 nM scrambled control siRNA for 72 h. The mRNA was isolated, and expression of Nox4 and Nox1 detected by qPCR. Graphs ( A and B ) show mean + SEM of Nox4 mRNA ( A ) or Nox1 mRNA ( B ) level relating to 18S mRNA. Data were normalized by setting scrambled control to 100% (*** P

    Journal: Cardiovascular Research

    Article Title: Resveratrol blocks Akt activation in angiotensin II- or EGF-stimulated vascular smooth muscle cells in a redox-independent manner

    doi: 10.1093/cvr/cvq355

    Figure Lengend Snippet: Ang II- and EGF-induced Akt phosphorylation and its inhibition by RV occur independent of Nox4. Knock-down of Nox4 was achieved by treating cells with 50 nM small interfering RNA (siRNA) against Nox4 or 50 nM scrambled control siRNA for 72 h. The mRNA was isolated, and expression of Nox4 and Nox1 detected by qPCR. Graphs ( A and B ) show mean + SEM of Nox4 mRNA ( A ) or Nox1 mRNA ( B ) level relating to 18S mRNA. Data were normalized by setting scrambled control to 100% (*** P

    Article Snippet: 2.6 Transfection with small interfering RNA and quantitative real-time PCR Five hours after seeding, VSMCs were transfected with small interfering RNA (siRNA; siNox4 from ThermoFisher Scientific, 50 nM; or scrambled siRNA control from Invitrogen) using Oligofectamine (Invitrogen) and Opti-MEM 1 (Gibco).

    Techniques: Inhibition, Small Interfering RNA, Isolation, Expressing, Real-time Polymerase Chain Reaction

    Requirement of ER stress for efficient brown adipogenesis. ( A and B ) HB2 brown preadipocytes were treated with or without 5 mM of 4-PBA or 100 μM of capsaicin or both for 8 days in the presence of insulin (20 nM). ( C and D ) HB2 brown preadipocytes were transfected with scrambled RNA or siRNA for Xbp1. Cells were treated with or without 100 μM of capsaicin in the presence of insulin (20 nM) for 8 days. ( A and C ) Oil Red O staining of cells on day 8 was performed, and the dye intensity was quantified (n = 2). ( B and D ) The expression level of Ucp1 was examined by RT-qPCR analysis. Black bar: vehicle; Hatched bar: capsaicin. The data are presented as the mean ± SE (n = 4). * P

    Journal: Scientific Reports

    Article Title: Supra-pharmacological concentration of capsaicin stimulates brown adipogenesis through induction of endoplasmic reticulum stress

    doi: 10.1038/s41598-018-19223-2

    Figure Lengend Snippet: Requirement of ER stress for efficient brown adipogenesis. ( A and B ) HB2 brown preadipocytes were treated with or without 5 mM of 4-PBA or 100 μM of capsaicin or both for 8 days in the presence of insulin (20 nM). ( C and D ) HB2 brown preadipocytes were transfected with scrambled RNA or siRNA for Xbp1. Cells were treated with or without 100 μM of capsaicin in the presence of insulin (20 nM) for 8 days. ( A and C ) Oil Red O staining of cells on day 8 was performed, and the dye intensity was quantified (n = 2). ( B and D ) The expression level of Ucp1 was examined by RT-qPCR analysis. Black bar: vehicle; Hatched bar: capsaicin. The data are presented as the mean ± SE (n = 4). * P

    Article Snippet: At confluence (day -2), cells were transfected with 6 μl of Lipofectamine RNAi Max (Thermo Fisher, Waltham, MA, USA) and 60 pmol of siRNA for Xbp1 or scrambled RNA (MISSION siRNA; Sigma) for 2 days.

    Techniques: Transfection, Staining, Expressing, Quantitative RT-PCR

    The three core TS cell transcription factors Cdx2, Elf5 and Tead4 are insufficient to complete reprogramming to induce a stable TS cell phenotype in bulk culture. ( a ) iCdx2 cells, shown previously to be capable of placental contribution 18 , and iCdx2/iRaf cells were transfected with an Elf5-2A-Tead4-2A-Hygro R (E+T) or Hygro R -only control (vec) expression construct, subjected to the trans-differentiation protocol for 6 days on antibiotic-resistant MEFs, FACS-purified from MEFs and assessed for gatekeeper gene expression levels by RT–qPCR. Details of overexpressed genes resulting from the various conditions are given in red. Elf5 and Tead4 expression, in conjunction with Cdx2 activation, improves the activation of some of the remaining gatekeeper genes. Values are of three biological replicates and expressed as percent of TS cell expression levels combined of the two independent TS cell lines (mean±s.e.m.). ( b ) Activation of TS cell transcription factors in the same cells as in ( a ). Hyperactivation of Tcfap2c and Hand1 (in a ) indicate onset of terminal trophoblast differentiation. Values expressed as in a . ( c ) Dynamics of methylation reprogramming of these cells at the ES-DMRs identified by meDIP-seq and assessed by Sequenom analysis as in Fig. 4c . ( d ) Assessment of phenotypic stability of reprogrammed TSL (iCdx2) cells. Upon removal of the MEF layer, the tight epithelial colony shape that is characteristic of bona fide TS cells was rapidly lost, and cells failed to maintain the co-expression of Cdx2 and Elf5 that is required to preserve the proliferative, self-renewing TS cell state, and instead started to differentiate. Scale bars: 200 μm (phase contrast), 100 μm (immunofluorescence). ( e ) RNA-seq analysis of iCdx2 (5ECER4G20) and iCdx2/iRaf ES cells grown for 5 days on MEFs, and then replated for 3 days on gelatinized TC plastic. Note the instability of gene expression, and specifically the downregulation of critical TS cell genes such as Eomes , Elf5 , Esrrb and Sox2 upon transfer of reprogrammed TSL cells from MEFs onto TC plastic.

    Journal: Nature Communications

    Article Title: Epigenetic memory of the first cell fate decision prevents complete ES cell reprogramming into trophoblast

    doi: 10.1038/ncomms6538

    Figure Lengend Snippet: The three core TS cell transcription factors Cdx2, Elf5 and Tead4 are insufficient to complete reprogramming to induce a stable TS cell phenotype in bulk culture. ( a ) iCdx2 cells, shown previously to be capable of placental contribution 18 , and iCdx2/iRaf cells were transfected with an Elf5-2A-Tead4-2A-Hygro R (E+T) or Hygro R -only control (vec) expression construct, subjected to the trans-differentiation protocol for 6 days on antibiotic-resistant MEFs, FACS-purified from MEFs and assessed for gatekeeper gene expression levels by RT–qPCR. Details of overexpressed genes resulting from the various conditions are given in red. Elf5 and Tead4 expression, in conjunction with Cdx2 activation, improves the activation of some of the remaining gatekeeper genes. Values are of three biological replicates and expressed as percent of TS cell expression levels combined of the two independent TS cell lines (mean±s.e.m.). ( b ) Activation of TS cell transcription factors in the same cells as in ( a ). Hyperactivation of Tcfap2c and Hand1 (in a ) indicate onset of terminal trophoblast differentiation. Values expressed as in a . ( c ) Dynamics of methylation reprogramming of these cells at the ES-DMRs identified by meDIP-seq and assessed by Sequenom analysis as in Fig. 4c . ( d ) Assessment of phenotypic stability of reprogrammed TSL (iCdx2) cells. Upon removal of the MEF layer, the tight epithelial colony shape that is characteristic of bona fide TS cells was rapidly lost, and cells failed to maintain the co-expression of Cdx2 and Elf5 that is required to preserve the proliferative, self-renewing TS cell state, and instead started to differentiate. Scale bars: 200 μm (phase contrast), 100 μm (immunofluorescence). ( e ) RNA-seq analysis of iCdx2 (5ECER4G20) and iCdx2/iRaf ES cells grown for 5 days on MEFs, and then replated for 3 days on gelatinized TC plastic. Note the instability of gene expression, and specifically the downregulation of critical TS cell genes such as Eomes , Elf5 , Esrrb and Sox2 upon transfer of reprogrammed TSL cells from MEFs onto TC plastic.

    Article Snippet: Cells were FACS-purified to separate them from MEFs, and total RNA prepared using TRI reagent (Sigma T9424) followed by DNase treatment using the TURBO DNA-free kit (Life Technologies AM1907), according to the manufacturers’ instructions. mRNA was isolated from total DNA-free RNA (150–240 ng) using the Dynabeads mRNA purification kit (Life Technologies 61006) and prepared into an indexed, strand-specific library using the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre SSV21106) according to the manufacturers’ instructions.

    Techniques: Transfection, Expressing, Construct, FACS, Purification, Quantitative RT-PCR, Activation Assay, Methylation, Methylated DNA Immunoprecipitation, Immunofluorescence, RNA Sequencing Assay

    Selection strategy by epithelial morphology in the most efficient iCdx2/iRaf reprogramming model. ( a ) Phase contrast images of TSL clones selected on the basis of colony shape and epithelial morphology. After an initial 5-day reprogramming on MEFs, cells were passaged for 5 weeks on tissue culture plastic in TS cell conditions in the presence of 4HT, during which time colonies were picked and expanded. Clone heterogeneity is observed, with clones CRVT3 and CRVT6 exhibiting the most genuinely TS-like epithelial colony shape. Scale bar: 200 μm. ( b ) Expression analysis of a cohort of trophoblast stem cell and differentiation-associated genes, as well as the identified gatekeeper loci, in selected clones. After growing/expanding colonies in the presence of 4HT for 5 weeks, cells were seeded ±4HT and RNA harvested after 2 days. Tight epithelial morphology in clones CRVT3 and CRVT6 correlates with TS-like Eomes expression levels. Overall, however, differentiation-associated markers are vastly upregulated, and gatekeeper gene expression remains unbalanced. Lack of transcriptional activation of Elf5 , Map3k8 , Sh2d3c and Tead4 is evident. Values are of three replicates and expressed as mean±s.e.m. ( c ) Immunostaining on these clones for Cdx2 and Elf5. Frequent lack of activation or re-silencing of Elf5, correlated with a more pronounced tendency to differentiate, is observed. ( d ) Immunostaining for TS cell surface markers Cd40 and Plet1. Plet1 is a GPI-anchored membrane-associated protein in TS cells, but is redistributed towards a cytoplasmic localization in differentiating trophoblast cells (arrowhead). Despite the TS-like colony shape, reprogrammed cell clones are extremely variable for Cd40 and largely fail to express Plet1 on their cell surface in a TS-like pattern and intensity. All photographs in c , d were taken at precisely the same exposure settings. Scale bars: 100 μm.

    Journal: Nature Communications

    Article Title: Epigenetic memory of the first cell fate decision prevents complete ES cell reprogramming into trophoblast

    doi: 10.1038/ncomms6538

    Figure Lengend Snippet: Selection strategy by epithelial morphology in the most efficient iCdx2/iRaf reprogramming model. ( a ) Phase contrast images of TSL clones selected on the basis of colony shape and epithelial morphology. After an initial 5-day reprogramming on MEFs, cells were passaged for 5 weeks on tissue culture plastic in TS cell conditions in the presence of 4HT, during which time colonies were picked and expanded. Clone heterogeneity is observed, with clones CRVT3 and CRVT6 exhibiting the most genuinely TS-like epithelial colony shape. Scale bar: 200 μm. ( b ) Expression analysis of a cohort of trophoblast stem cell and differentiation-associated genes, as well as the identified gatekeeper loci, in selected clones. After growing/expanding colonies in the presence of 4HT for 5 weeks, cells were seeded ±4HT and RNA harvested after 2 days. Tight epithelial morphology in clones CRVT3 and CRVT6 correlates with TS-like Eomes expression levels. Overall, however, differentiation-associated markers are vastly upregulated, and gatekeeper gene expression remains unbalanced. Lack of transcriptional activation of Elf5 , Map3k8 , Sh2d3c and Tead4 is evident. Values are of three replicates and expressed as mean±s.e.m. ( c ) Immunostaining on these clones for Cdx2 and Elf5. Frequent lack of activation or re-silencing of Elf5, correlated with a more pronounced tendency to differentiate, is observed. ( d ) Immunostaining for TS cell surface markers Cd40 and Plet1. Plet1 is a GPI-anchored membrane-associated protein in TS cells, but is redistributed towards a cytoplasmic localization in differentiating trophoblast cells (arrowhead). Despite the TS-like colony shape, reprogrammed cell clones are extremely variable for Cd40 and largely fail to express Plet1 on their cell surface in a TS-like pattern and intensity. All photographs in c , d were taken at precisely the same exposure settings. Scale bars: 100 μm.

    Article Snippet: Cells were FACS-purified to separate them from MEFs, and total RNA prepared using TRI reagent (Sigma T9424) followed by DNase treatment using the TURBO DNA-free kit (Life Technologies AM1907), according to the manufacturers’ instructions. mRNA was isolated from total DNA-free RNA (150–240 ng) using the Dynabeads mRNA purification kit (Life Technologies 61006) and prepared into an indexed, strand-specific library using the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre SSV21106) according to the manufacturers’ instructions.

    Techniques: Selection, Clone Assay, Expressing, Activation Assay, Immunostaining

    Effect of cholesterol and phosphatidylcholine on the transcription of cholesterol transporters Fully confluent fibroblasts were incubated for 24 hours in DMEM (BSA) in the absence of lipid (control), the presence of cholesterol (20 μg/mL) alone (chol), or a combination of 20 μg/mL cholesterol and 100μg/mL phosphotidylcholine (chol+PC). Total RNA was extracted, and amplicons for ABCA1, ABCG1, SR-BI and β-actin were amplified by RT-PCR (25 cycles), using gene-specific primers ( Table 1 ). ( A ) PCR products were resolved on 2% agarose gels. ( B ) Data in bar graphs were obtained by quantifying the intensity of bands in gel images using Image J. The transporter to β-actin ratios were calculated and values for control cells was set to 100%. The data are representative of at least three experiments with similar results

    Journal: International journal of biochemistry research & review

    Article Title: Phosphatidylcholine-Mediated Aqueous Diffusion of Cellular Cholesterol Down-Regulates the ABCA1 Transporter in Human Skin Fibroblasts

    doi: 10.9734/IJBCRR/2015/14058

    Figure Lengend Snippet: Effect of cholesterol and phosphatidylcholine on the transcription of cholesterol transporters Fully confluent fibroblasts were incubated for 24 hours in DMEM (BSA) in the absence of lipid (control), the presence of cholesterol (20 μg/mL) alone (chol), or a combination of 20 μg/mL cholesterol and 100μg/mL phosphotidylcholine (chol+PC). Total RNA was extracted, and amplicons for ABCA1, ABCG1, SR-BI and β-actin were amplified by RT-PCR (25 cycles), using gene-specific primers ( Table 1 ). ( A ) PCR products were resolved on 2% agarose gels. ( B ) Data in bar graphs were obtained by quantifying the intensity of bands in gel images using Image J. The transporter to β-actin ratios were calculated and values for control cells was set to 100%. The data are representative of at least three experiments with similar results

    Article Snippet: 2.2 RNA Isolation and RT-PCR RNA was isolated using TRI reagent (Sigma) according to the manufacturer’s protocol.

    Techniques: Incubation, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Relationship between SDV and RIN . Comparison of the SDV and RIN integrity metrics for a panel of seven RNA samples.

    Journal: BMC Research Notes

    Article Title: Evaluation of a novel approach for the measurement of RNA quality

    doi: 10.1186/1756-0500-3-89

    Figure Lengend Snippet: Relationship between SDV and RIN . Comparison of the SDV and RIN integrity metrics for a panel of seven RNA samples. "Treatment" denotes the duration of thermal degradation treatment used. Each group includes nine observations, except for treatment four which corresponds to a 12-minute incubation at 90°C and, which includes only seven observations. The thicker horizontal line shows the median, boxes show the upper and lower quartiles and whiskers extend to the most distant data point within 1.5 times the interquartile range of the relevant quartile. Values beyond this are shown as individual data points.

    Article Snippet: Comparison with RNA Integrity Number (RIN) RIN is an incremental scale which spans from 0 to 10, with increasing RNA integrity correlating with increasing RIN value.

    Techniques: Incubation

    SDV and RIN chromatograms . Graphical overlay of SDV chromatograms for the analysis of intact (A), and degraded (B) RNA. RIN chromatograms for intact (C) and degraded (D) RNA are shown for comparison.

    Journal: BMC Research Notes

    Article Title: Evaluation of a novel approach for the measurement of RNA quality

    doi: 10.1186/1756-0500-3-89

    Figure Lengend Snippet: SDV and RIN chromatograms . Graphical overlay of SDV chromatograms for the analysis of intact (A), and degraded (B) RNA. RIN chromatograms for intact (C) and degraded (D) RNA are shown for comparison.

    Article Snippet: Comparison with RNA Integrity Number (RIN) RIN is an incremental scale which spans from 0 to 10, with increasing RNA integrity correlating with increasing RIN value.

    Techniques:

    Amplification of a serial dilution of the EUROHEP standard with HBV-specific primers in a nested PCR following extraction with the Chemagic DNA/RNA kit and the QIAamp DNA Blood Mini kit. (A) Chemagen extracts. (B) QIAGEN extracts. Lane M shows a 100-bp ladder, and lane C shows results for the negative control. Lanes 1 through 10 show results for different viral loads expressed in numbers of copies per ml: 1, 4 × 10 5 ; 2, 4 × 10 4 ; 3, 4 × 10 3 ; 4, 4 × 10 2 ; 5, 2 × 10 2 ; 6, 1 × 10 2 ; 7, 8 × 10 1 ; 8, 4 × 10 1 ; 9, 2 × 10 1 ; 10, 1 × 10 1 .

    Journal: Journal of Clinical Microbiology

    Article Title: Efficient Extraction of Viral DNA and Viral RNA by the Chemagic Viral DNA/RNA Kit Allows Sensitive Detection of Cytomegalovirus, Hepatitis B Virus, and Hepatitis G Virus by PCR

    doi: 10.1128/JCM.41.11.5273-5276.2003

    Figure Lengend Snippet: Amplification of a serial dilution of the EUROHEP standard with HBV-specific primers in a nested PCR following extraction with the Chemagic DNA/RNA kit and the QIAamp DNA Blood Mini kit. (A) Chemagen extracts. (B) QIAGEN extracts. Lane M shows a 100-bp ladder, and lane C shows results for the negative control. Lanes 1 through 10 show results for different viral loads expressed in numbers of copies per ml: 1, 4 × 10 5 ; 2, 4 × 10 4 ; 3, 4 × 10 3 ; 4, 4 × 10 2 ; 5, 2 × 10 2 ; 6, 1 × 10 2 ; 7, 8 × 10 1 ; 8, 4 × 10 1 ; 9, 2 × 10 1 ; 10, 1 × 10 1 .

    Article Snippet: Identical results were obtained after extraction of the identical specimens with the QIAamp Viral RNA Mini kit.

    Techniques: Amplification, Serial Dilution, Nested PCR, Negative Control

    Amplification of a serial dilution of the standard plasmid pCR-gpB by real-time PCR with CMV-specific primers (amplicon, 254 bp) on the LightCycler instrument (software, version 3.5; analysis method, fit points with manual noise band adjustment, hybridization probe format) following extraction with the Chemagic DNA/RNA kit and the QIAamp DNA Blood Mini kit. pCR-gpB concentrations (from left to right) were 2 × 10 5 , 2 × 10 4 , 2 × 10 3 , and 2 × 10 2 copies per ml of serum. Continuous line, Chemagen extracts; broken line, QIAGEN extracts. Cycle number, cycle number of the amplification reaction; F2/F1, quotient of fluorescence channel F2 (hybridization probe) and fluorescence channel F1 (fluorescein).

    Journal: Journal of Clinical Microbiology

    Article Title: Efficient Extraction of Viral DNA and Viral RNA by the Chemagic Viral DNA/RNA Kit Allows Sensitive Detection of Cytomegalovirus, Hepatitis B Virus, and Hepatitis G Virus by PCR

    doi: 10.1128/JCM.41.11.5273-5276.2003

    Figure Lengend Snippet: Amplification of a serial dilution of the standard plasmid pCR-gpB by real-time PCR with CMV-specific primers (amplicon, 254 bp) on the LightCycler instrument (software, version 3.5; analysis method, fit points with manual noise band adjustment, hybridization probe format) following extraction with the Chemagic DNA/RNA kit and the QIAamp DNA Blood Mini kit. pCR-gpB concentrations (from left to right) were 2 × 10 5 , 2 × 10 4 , 2 × 10 3 , and 2 × 10 2 copies per ml of serum. Continuous line, Chemagen extracts; broken line, QIAGEN extracts. Cycle number, cycle number of the amplification reaction; F2/F1, quotient of fluorescence channel F2 (hybridization probe) and fluorescence channel F1 (fluorescein).

    Article Snippet: Identical results were obtained after extraction of the identical specimens with the QIAamp Viral RNA Mini kit.

    Techniques: Amplification, Serial Dilution, Plasmid Preparation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Software, Hybridization, Fluorescence

    IL-10 mRNA expression by B10 cells after LPS and/or CD40L stimulation. Cultured mouse slpenocytes were treated with E. coli LPS (10 µg/ml) and/or CD40L (1 µg/ml) for 2 days and CD19 + CD1d hi CD5 + cells in the cultured splenocytes were sorted out using flow cytometry. Total RNA were isolated from sorted cells using a Purelink RNA mini kit (Life Technology) and IL-10 expression was determined by real time PCR. At least 100,000 cells were sorted from each sample for PCR. Data are presented as mean ± SEM, n = 6. Student t-test, *P

    Journal: Open journal of immunology

    Article Title: Lipopolysaccharide Attenuates CD40 Ligand-Induced Regulatory B10 Cell Expansion and IL-10 Production in Mouse Splenocytes

    doi: 10.4236/oji.2015.51001

    Figure Lengend Snippet: IL-10 mRNA expression by B10 cells after LPS and/or CD40L stimulation. Cultured mouse slpenocytes were treated with E. coli LPS (10 µg/ml) and/or CD40L (1 µg/ml) for 2 days and CD19 + CD1d hi CD5 + cells in the cultured splenocytes were sorted out using flow cytometry. Total RNA were isolated from sorted cells using a Purelink RNA mini kit (Life Technology) and IL-10 expression was determined by real time PCR. At least 100,000 cells were sorted from each sample for PCR. Data are presented as mean ± SEM, n = 6. Student t-test, *P

    Article Snippet: Reverse Transcription and Quantitative Real Time PCR Total RNA was extracted from the cells using a Purelink RNA mini kit (Life Technology) following manufacturer’s instructions.

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

    Sequence alignment of HIV vmiRs with consensus genomic sequence from HIV-1 subtypes and absolute quantitation of miRNAs by Real Time RT-PCR. A : Alignment of vmiR sequences of GU-rich tract is consistent with consensus genomic sequence from HIV-1 subtypes A-J, 533 isolates. B : VmiR99 is 90–100% identical to 96% of HIV-1 sequences. Sequences within the GU tract, vmiR88, vmiR99 and ssRNA40 were aligned with 196, 201, 254 and 272 genome sequences, respectively. C : Genomic RNA of HIV-1 BaL strain was scanned for every 21-bp RNA segment and the distribution of base compositions (46.5±11.8% G+U) is shown. VmiR-TAR is GU-poor (35%). VmiR88 and vmiR99 are GU-rich (71% and 76% G+U, respectively). Absolute quantitation of miRNAs was determined by Real Time RT-PCR. After first strand cDNA synthesis, amplification ( D, F, H ) and absolute quantitation ( E, G, I ) of vmiR-TAR ( D–E ) vmiR88 ( F–G ), vmiR99 ( H–I ) and RNA40 (I) was standardized using synthetic miR oligonucleotides in the miRCURY LNA Universal RT microRNA PCR method (Exiqon) on an ABI 7900HT FAST Real Time PCR system. ΔR n is the change in normalized reporter fluorescence intensity. C T is the threshold cycle in which the amplification curve crosses the dashed horizontal line. Data depict a representative experiment done in duplicate.

    Journal: PLoS ONE

    Article Title: Novel HIV-1 MiRNAs Stimulate TNFα Release in Human Macrophages via TLR8 Signaling Pathway

    doi: 10.1371/journal.pone.0106006

    Figure Lengend Snippet: Sequence alignment of HIV vmiRs with consensus genomic sequence from HIV-1 subtypes and absolute quantitation of miRNAs by Real Time RT-PCR. A : Alignment of vmiR sequences of GU-rich tract is consistent with consensus genomic sequence from HIV-1 subtypes A-J, 533 isolates. B : VmiR99 is 90–100% identical to 96% of HIV-1 sequences. Sequences within the GU tract, vmiR88, vmiR99 and ssRNA40 were aligned with 196, 201, 254 and 272 genome sequences, respectively. C : Genomic RNA of HIV-1 BaL strain was scanned for every 21-bp RNA segment and the distribution of base compositions (46.5±11.8% G+U) is shown. VmiR-TAR is GU-poor (35%). VmiR88 and vmiR99 are GU-rich (71% and 76% G+U, respectively). Absolute quantitation of miRNAs was determined by Real Time RT-PCR. After first strand cDNA synthesis, amplification ( D, F, H ) and absolute quantitation ( E, G, I ) of vmiR-TAR ( D–E ) vmiR88 ( F–G ), vmiR99 ( H–I ) and RNA40 (I) was standardized using synthetic miR oligonucleotides in the miRCURY LNA Universal RT microRNA PCR method (Exiqon) on an ABI 7900HT FAST Real Time PCR system. ΔR n is the change in normalized reporter fluorescence intensity. C T is the threshold cycle in which the amplification curve crosses the dashed horizontal line. Data depict a representative experiment done in duplicate.

    Article Snippet: First strand cDNA was synthesized with the High Capacity RNA-to-cDNA kit (Applied Biosystems) using a GeneAmp PCR System 9600 (Perkin Elmer) set for 37°C for 60 min, 95°C for 5 min followed by 4°C.

    Techniques: Sequencing, Quantitation Assay, Quantitative RT-PCR, Amplification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Fluorescence

    RNA quality of cultivated tissue slices. RNA quality was determined by a Bioanalyzer 2100 using the RNA 6000 Nano-Kit (Agilent Technologies) and revealed good quality before the DNase digestion was performed ( a ). After the DNase digestion, the RNA quality was strongly reduced ( b ). The left graphs show untreated peritumoral brain tissue, the right graphs the corresponding GBM tissue.

    Journal: Scientific Reports

    Article Title: Deep sequencing and automated histochemistry of human tissue slice cultures improve their usability as preclinical model for cancer research

    doi: 10.1038/s41598-019-56509-5

    Figure Lengend Snippet: RNA quality of cultivated tissue slices. RNA quality was determined by a Bioanalyzer 2100 using the RNA 6000 Nano-Kit (Agilent Technologies) and revealed good quality before the DNase digestion was performed ( a ). After the DNase digestion, the RNA quality was strongly reduced ( b ). The left graphs show untreated peritumoral brain tissue, the right graphs the corresponding GBM tissue.

    Article Snippet: RNA quality was determined by the Bioanalyzer 2100 using the RNA 6000 Nano-Kit (Agilent Technologies).

    Techniques:

    Gene body H3K36me3 level is negatively correlated with age-dependent mRNA expression change in Drosophila heads. ( A ) All genes with mappable H3K36me3 ChIP–chip and RNA-seq reads are plotted in the order of normalized H3K36me3 levels at the young

    Journal: Genes & Development

    Article Title: Trimethylation of Lys36 on H3 restricts gene expression change during aging and impacts life span

    doi: 10.1101/gad.254144.114

    Figure Lengend Snippet: Gene body H3K36me3 level is negatively correlated with age-dependent mRNA expression change in Drosophila heads. ( A ) All genes with mappable H3K36me3 ChIP–chip and RNA-seq reads are plotted in the order of normalized H3K36me3 levels at the young

    Article Snippet: Two-hundred nanograms of mRNA was then used to prepare RNA-seq libraries, which were sequenced on an Illumina HiSeq 2000 at six samples per lane.

    Techniques: Expressing, Chromatin Immunoprecipitation, RNA Sequencing Assay

    VFP4-controlled genes ATL31 and At2g32030 . Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of ATL31 and At2g32030 expression in roots of A , wild-type Col-0 and vfp4-1 plants and B , wild-type Col-0 and VFP4 OE-6 plants. C , RT-qPCR analysis of ATL31 and At2g32030 expression in roots of the wild-type Col-0, ATL31 OE-4, and ATL31 OE-5 and Col-0 and At2g32030 OE-9 plants, respectively. The levels of expression were normalized to the internal reference genes ACT7 and 18S RNA . The expression level of each tested gene in the wild-type Col-0 is set to 1.0, and error bars represent standard error of the mean of independent biological replicates, n = 3.

    Journal: Molecular plant-microbe interactions : MPMI

    Article Title: The Agrobacterium F-Box Protein Effector VirF Destabilizes the Arabidopsis GLABROUS1 Enhancer/Binding Protein-Like Transcription Factor VFP4, a Transcriptional Activator of Defense Response Genes

    doi: 10.1094/MPMI-07-17-0188-FI

    Figure Lengend Snippet: VFP4-controlled genes ATL31 and At2g32030 . Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of ATL31 and At2g32030 expression in roots of A , wild-type Col-0 and vfp4-1 plants and B , wild-type Col-0 and VFP4 OE-6 plants. C , RT-qPCR analysis of ATL31 and At2g32030 expression in roots of the wild-type Col-0, ATL31 OE-4, and ATL31 OE-5 and Col-0 and At2g32030 OE-9 plants, respectively. The levels of expression were normalized to the internal reference genes ACT7 and 18S RNA . The expression level of each tested gene in the wild-type Col-0 is set to 1.0, and error bars represent standard error of the mean of independent biological replicates, n = 3.

    Article Snippet: For RT-PCR analysis of VFP4 expression in vfp4-1 plants, total RNA was extracted from leaves of the wild-type Col-0 and vfp4-1 plants using TRIzol (Invitrogen) and was purified with the SV total RNA isolation system (Promega).

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

    VFP4 localizes to the nucleus and cytoplasm. A , Subcellular localization of VFP4. Location of the cell nucleus is indicated by a white arrowhead. All images are projections of single confocal sections and are representative images of three independent experiments. Scale bar = 20 μm. B , Real-time quantitative polymerase chain reaction analysis of VFP4 gene expression following inoculation of wild-type plants by agrobacterium. The levels of expression were normalized to those of ACT7 and 18S RNA . The expression level of VFP4 in the mock-inoculated plants is set to 1.0, and error bars represent standard error of the mean of independent biological replicates, n = 3.

    Journal: Molecular plant-microbe interactions : MPMI

    Article Title: The Agrobacterium F-Box Protein Effector VirF Destabilizes the Arabidopsis GLABROUS1 Enhancer/Binding Protein-Like Transcription Factor VFP4, a Transcriptional Activator of Defense Response Genes

    doi: 10.1094/MPMI-07-17-0188-FI

    Figure Lengend Snippet: VFP4 localizes to the nucleus and cytoplasm. A , Subcellular localization of VFP4. Location of the cell nucleus is indicated by a white arrowhead. All images are projections of single confocal sections and are representative images of three independent experiments. Scale bar = 20 μm. B , Real-time quantitative polymerase chain reaction analysis of VFP4 gene expression following inoculation of wild-type plants by agrobacterium. The levels of expression were normalized to those of ACT7 and 18S RNA . The expression level of VFP4 in the mock-inoculated plants is set to 1.0, and error bars represent standard error of the mean of independent biological replicates, n = 3.

    Article Snippet: For RT-PCR analysis of VFP4 expression in vfp4-1 plants, total RNA was extracted from leaves of the wild-type Col-0 and vfp4-1 plants using TRIzol (Invitrogen) and was purified with the SV total RNA isolation system (Promega).

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Gain-of-function VFP4 OE-6 and VFP4 OE-14 plants exhibit reduced susceptibility to Agrobacterium tumorigenicity. A , Real-time quantitative polymerase chain reaction analysis of VFP4 gene expression in roots of wild-type Col-0, VFP4 OE-6, and VFP4 OE-14 plants. The levels of expression were normalized to the internal reference genes ACT7 and 18S RNA . The expression level of VFP4 in the wild-type Col-0 was set to 1.0, and error bars represent standard error of the mean (SEM) of independent biological replicates, n = 3. B , Tumors developed on root explants inoculated with A. tumefaciens . C , Quantification of tumorigenicity. Error bars represent SEM of n = 3 biological replicates. Differences in tumorigenicity values between wild-type Col-0 and VFP4 OE-14 plants indicated by different letters are statistically significant ( P values

    Journal: Molecular plant-microbe interactions : MPMI

    Article Title: The Agrobacterium F-Box Protein Effector VirF Destabilizes the Arabidopsis GLABROUS1 Enhancer/Binding Protein-Like Transcription Factor VFP4, a Transcriptional Activator of Defense Response Genes

    doi: 10.1094/MPMI-07-17-0188-FI

    Figure Lengend Snippet: Gain-of-function VFP4 OE-6 and VFP4 OE-14 plants exhibit reduced susceptibility to Agrobacterium tumorigenicity. A , Real-time quantitative polymerase chain reaction analysis of VFP4 gene expression in roots of wild-type Col-0, VFP4 OE-6, and VFP4 OE-14 plants. The levels of expression were normalized to the internal reference genes ACT7 and 18S RNA . The expression level of VFP4 in the wild-type Col-0 was set to 1.0, and error bars represent standard error of the mean (SEM) of independent biological replicates, n = 3. B , Tumors developed on root explants inoculated with A. tumefaciens . C , Quantification of tumorigenicity. Error bars represent SEM of n = 3 biological replicates. Differences in tumorigenicity values between wild-type Col-0 and VFP4 OE-14 plants indicated by different letters are statistically significant ( P values

    Article Snippet: For RT-PCR analysis of VFP4 expression in vfp4-1 plants, total RNA was extracted from leaves of the wild-type Col-0 and vfp4-1 plants using TRIzol (Invitrogen) and was purified with the SV total RNA isolation system (Promega).

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Dominant-negative effect of L-protein mutants. (A) Inactive mutants were transfected in a ratio of 1:1 with wild-type L protein. As a control, a PCR fragment of the L gene lacking the upstream T7 RNA polymerase promoter was transfected 1:1 with the complete

    Journal: Journal of Virology

    Article Title: Mutational Evidence for a Structural Model of the Lassa Virus RNA Polymerase Domain and Identification of Two Residues, Gly1394 and Asp1395, That Are Critical for Transcription but Not Replication of the Genome ▿

    doi: 10.1128/JVI.00220-08

    Figure Lengend Snippet: Dominant-negative effect of L-protein mutants. (A) Inactive mutants were transfected in a ratio of 1:1 with wild-type L protein. As a control, a PCR fragment of the L gene lacking the upstream T7 RNA polymerase promoter was transfected 1:1 with the complete

    Article Snippet: BSR T7/5 cells ( ) stably expressing T7 RNA polymerase and BHK-21 cells were grown in Glasgow modified Eagle medium (MEM; Gibco) supplemented with 5% fetal calf serum (FCS).

    Techniques: Dominant Negative Mutation, Transfection, Polymerase Chain Reaction

    Validation of gene expression changes estimated with CANEapp with quantitative real-time PCR. a RNA-seq analysis of hippocampi of Alzheimer’s disease patients and controls. Hippocampal tissue from 4 AD patients and 4 control individuals was used to extract total RNA and perform ribodepletion and strand-specific library preparation. Single-end RNA sequencing was performed on Illumina HiSeq 2000. Fold changes of expression for 2 downregulated and 4 upregulated genes measured with real-time PCR was compared with expression values generated by CANEapp. b RNA-seq of developing mouse cortex. Tissue from 4 embryonic day 17 and 3 adult mouse cortical samples was processed to extract polyA-selected RNA and generate paired-end unidirectional sequencing data with Illumina Genome Analyzer IIx. Gene expression estimates of 4 downregulated and 4 upregulated genes were compared between CANEapp and real-time PCR. c Fold changes of gene expression for RNA-seq of liver of rats treated with two DNA-damage compounds. The data was produced by paired-end sequencing of polyA-selected RNA on Illumina HiSeq 2000. Fold changes of expression for 2 downregulated and 4 upregulated genes were compared between CANEapp and real-time PCR. R 2 -coefficient of determination

    Journal: BMC Genomics

    Article Title: CANEapp: a user-friendly application for automated next generation transcriptomic data analysis

    doi: 10.1186/s12864-015-2346-y

    Figure Lengend Snippet: Validation of gene expression changes estimated with CANEapp with quantitative real-time PCR. a RNA-seq analysis of hippocampi of Alzheimer’s disease patients and controls. Hippocampal tissue from 4 AD patients and 4 control individuals was used to extract total RNA and perform ribodepletion and strand-specific library preparation. Single-end RNA sequencing was performed on Illumina HiSeq 2000. Fold changes of expression for 2 downregulated and 4 upregulated genes measured with real-time PCR was compared with expression values generated by CANEapp. b RNA-seq of developing mouse cortex. Tissue from 4 embryonic day 17 and 3 adult mouse cortical samples was processed to extract polyA-selected RNA and generate paired-end unidirectional sequencing data with Illumina Genome Analyzer IIx. Gene expression estimates of 4 downregulated and 4 upregulated genes were compared between CANEapp and real-time PCR. c Fold changes of gene expression for RNA-seq of liver of rats treated with two DNA-damage compounds. The data was produced by paired-end sequencing of polyA-selected RNA on Illumina HiSeq 2000. Fold changes of expression for 2 downregulated and 4 upregulated genes were compared between CANEapp and real-time PCR. R 2 -coefficient of determination

    Article Snippet: RNA in human and rat experiments was sequenced on the Illumina HiSeq 2000 machine, whereas mouse RNA-seq data was produced by sequencing RNA on Illumina GA-IIx sequencer.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Generated, Sequencing, Produced

    NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p

    Journal: Cell stem cell

    Article Title: Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration

    doi: 10.1016/j.stem.2016.12.015

    Figure Lengend Snippet: NAM effectively reduces production of complement factors (A) Altered expression of several genes in the complement pathway detected by RNA-seq analysis of NAM treatment (n=7 donors; 4 AMD and 3 control; 7 lines). ; each sample is color matched across NAM and vehicle treatment. (C) ELISA measurement of secretion of C3 into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (D) qPCR analysis of AMD/drusen associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (E) ELISA measurement of secretion of VEGF-A and APOJ into the culture supernatant 60–72 hours after the last medium change in vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM of absorbance from ELISA assay (n=3 donors; 2 AMD and 1 control; 4 lines). (F) qPCR analysis of complement and inflammation associated protein transcripts in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (G) LDH release into the culture supernatant of vehicle, 10mM NAM and C3shRNA treated hiPSC-RPE. Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). (H) qPCR analysis of TP53 and RPE genes expression in C3shRNA and 10mM NAM treated hiPSC-RPE relative to vehicle (baseline defined as 100%). Data are expressed as mean± SEM (n=3 donors; 2 AMD and 1 control; 4 lines). Paired Student’s t test (two-tailed) was used for statistical analysis. Paired Student’s t test (one-tailed) was used for statistical analysis, unless stated otherwise above (*= p

    Article Snippet: To extract RNA, hiPSC-RPE cells were scraped from the culture wells and collected in RNA protect (Cat. # 76526; Qiagen).

    Techniques: Expressing, RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, One-tailed Test

    RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .

    Journal: Cell stem cell

    Article Title: Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-related Macular Degeneration

    doi: 10.1016/j.stem.2016.12.015

    Figure Lengend Snippet: RNA-seq analysis of action of NAM on hiPSC-RPE (A) Gene interaction network (confidence level=0.9) of the differentially expressed genes in NAM treated hiPSC-RPE compared to vehicle from RNA-seq analysis, using the STRING database (n=7 donors; 4 AMD and 3 control; 7 lines). Subnetworks (Neighborhoods) are colored and annotated with enriched functional categories. Gray lines: connections within a neighborhood; Red lines: connections between neighborhoods; Squares: Upregulated genes; Circles: Downregulated genes. (B-C) GO enrichment for KEGG pathways (B) and disease associations (C) of the differentially expressed genes (n=7 donors; 4 AMD and 3 control; 7 lines). .

    Article Snippet: To extract RNA, hiPSC-RPE cells were scraped from the culture wells and collected in RNA protect (Cat. # 76526; Qiagen).

    Techniques: RNA Sequencing Assay, Functional Assay

    HCMV infection leads to an upregulation of polycomb group proteins. (A and B) HFF cells were infected with the laboratory HCMV strain AD169 (A) and the clinical strain TB40/E (B) at an MOI of 3. Samples were harvested at the indicated time points postinfection, and whole-cell extracts were subjected to subsequent SDS-PAGE and Western blot analysis. Cellular PcG proteins were detected by the indicated antibodies. The detection of viral immediate early (IE1), early (pUL44), and late (pp28, MCP) proteins was used to monitor progression of the replication cycle. β-Actin served as a loading control. (C) HFF cells were infected with AD169 at an MOI of 3 and harvested at the indicated time points postinfection. RNA was isolated using TRIzol and the Direct-zol RNA miniprep kit (Zymo Research) and synthesized into cDNA by RT-PCR. Transcript levels were assessed by SYBR green PCR, and relative mRNA levels were calculated by normalization against the value for the housekeeping gene GAPDH. Values are derived from biological triplicates and represent mean values ± standard deviations (SD). Statistical analysis was performed by ordinary one-way analysis of variance (ANOVA). n.s., not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (D) HFF cells were infected with AD169 at an MOI of 3 and treated with cycloheximide (CHX) (150 μg/ml) in parallel. After the indicated time points postinfection, whole-cell lysates were prepared and subjected to Western blot analysis. Cellular and viral proteins were detected by the indicated antibodies. IE proteins (IE1 or IE2) served as controls for blocked de novo protein synthesis, while pp65 ensured the income of tegument proteins. β-Actin served as a loading control. (E) HFF cells were infected with either AD169 or UV-inactivated AD169 at an MOI of 1. Whole-cell extracts were generated at the indicated time points and subjected to Western blot analysis. Detection of proteins was equivalent to that for panel D.

    Journal: Journal of Virology

    Article Title: A Noncanonical Function of Polycomb Repressive Complexes Promotes Human Cytomegalovirus Lytic DNA Replication and Serves as a Novel Cellular Target for Antiviral Intervention

    doi: 10.1128/JVI.02143-18

    Figure Lengend Snippet: HCMV infection leads to an upregulation of polycomb group proteins. (A and B) HFF cells were infected with the laboratory HCMV strain AD169 (A) and the clinical strain TB40/E (B) at an MOI of 3. Samples were harvested at the indicated time points postinfection, and whole-cell extracts were subjected to subsequent SDS-PAGE and Western blot analysis. Cellular PcG proteins were detected by the indicated antibodies. The detection of viral immediate early (IE1), early (pUL44), and late (pp28, MCP) proteins was used to monitor progression of the replication cycle. β-Actin served as a loading control. (C) HFF cells were infected with AD169 at an MOI of 3 and harvested at the indicated time points postinfection. RNA was isolated using TRIzol and the Direct-zol RNA miniprep kit (Zymo Research) and synthesized into cDNA by RT-PCR. Transcript levels were assessed by SYBR green PCR, and relative mRNA levels were calculated by normalization against the value for the housekeeping gene GAPDH. Values are derived from biological triplicates and represent mean values ± standard deviations (SD). Statistical analysis was performed by ordinary one-way analysis of variance (ANOVA). n.s., not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (D) HFF cells were infected with AD169 at an MOI of 3 and treated with cycloheximide (CHX) (150 μg/ml) in parallel. After the indicated time points postinfection, whole-cell lysates were prepared and subjected to Western blot analysis. Cellular and viral proteins were detected by the indicated antibodies. IE proteins (IE1 or IE2) served as controls for blocked de novo protein synthesis, while pp65 ensured the income of tegument proteins. β-Actin served as a loading control. (E) HFF cells were infected with either AD169 or UV-inactivated AD169 at an MOI of 1. Whole-cell extracts were generated at the indicated time points and subjected to Western blot analysis. Detection of proteins was equivalent to that for panel D.

    Article Snippet: RNAs from 6 × 105 mock-infected or infected HFF cells were isolated in triplicate using TRIzol reagent (Invitrogen, Karlsruhe, Germany) and a Direct-zol RNA miniprep kit (Zymo Research, Freiburg, Germany) according to the manufacturers’ instructions.

    Techniques: Infection, SDS Page, Western Blot, Isolation, Synthesized, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction, Derivative Assay, Generated

    A scatterplot showing the ratio of live, potentially active bacteria (RNA/DNA) in tumor vs . non-tumor tissue.

    Journal: PLoS ONE

    Article Title: Characterization of the fecal and mucosa-associated microbiota in dogs with colorectal epithelial tumors

    doi: 10.1371/journal.pone.0198342

    Figure Lengend Snippet: A scatterplot showing the ratio of live, potentially active bacteria (RNA/DNA) in tumor vs . non-tumor tissue.

    Article Snippet: Isolation of DNA and RNA from mucosal samples and cDNA synthesis Using the AllPrep DNA/RNA Mini Kit (Qiagen), RNA and DNA were isolated from ~ 8 mg of mucosal tissue that had been preserved in Allprotect Tissue Reagent (Qiagen).

    Techniques:

    A codon-modified GAr sequence influences EBNA1 synthesis. ( A ) IVT assay of pcDNA3 expression constructs encoding EBNA1 (E1) (lane 1), E1ΔGA (lane 2), E1-GAr(100N) (lane 3), E1-GAr(100M) (lane 4), E1-GAr(200N) (lane 5), E1-GAr(200M) (lane 6), E1-GAr(300N) (lane 7), E1-GAr(300M) (lane 8), E1-GAr(400N) (lane 9), E1-GAr(400M) (lane 10), E1-GAr(500N) (lane 11), or E1-GAr(500M) (lane 12). The constructs were transcribed and translated in vitro with T7 RNA polymerase by using a coupled transcription/translation reticulocyte lysate system. 35 S-methionine-labeled proteins were visualized by autoradiography. ( B and C ) Band intensities from the IVT assay were quantified by densitometric analysis using Imagequant software (Molecular Dynamics) and graphed to demonstrate absolute intensities ( B ) or relative fold increase of EBNA1 encoded by codon-modified GAr domains compared with EBNA1 encoded by native GAr domains ( C ). ( D ) Western blot of EBV-negative HEK293 cells transfected with expression constructs encoding E1-GFP (lane 1), E1ΔGA-GFP (lane 2), E1-GAr(100N)-GFP (lane 3), E1-GAr(100M)-GFP (lane 4), E1-GAr(200N)-GFP (lane 5), E1-GAr(200M)-GFP (lane 6), E1-GAr(300N)-GFP (lane 7), E1-GAr(300M)-GFP (lane 8), E1-GAr(400N)-GFP (lane 9), E1-GAr(400M)-GFP (lane 10), E1-GAr(500N)-GFP (lane 11), or E1-GAr(500M)-GFP (lane 12) with a GFP antibody ( Upper ) or a monoclonal actin antibody ( Lower ). Molecular weight markers M r (kDa) are indicated on the left. ( E ) Band intensities after immunoblotting were quantified as described for B . Representative data from one of four experiments are presented here.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of protein translation through mRNA structure influences MHC class I loading and T cell recognition

    doi: 10.1073/pnas.0801968105

    Figure Lengend Snippet: A codon-modified GAr sequence influences EBNA1 synthesis. ( A ) IVT assay of pcDNA3 expression constructs encoding EBNA1 (E1) (lane 1), E1ΔGA (lane 2), E1-GAr(100N) (lane 3), E1-GAr(100M) (lane 4), E1-GAr(200N) (lane 5), E1-GAr(200M) (lane 6), E1-GAr(300N) (lane 7), E1-GAr(300M) (lane 8), E1-GAr(400N) (lane 9), E1-GAr(400M) (lane 10), E1-GAr(500N) (lane 11), or E1-GAr(500M) (lane 12). The constructs were transcribed and translated in vitro with T7 RNA polymerase by using a coupled transcription/translation reticulocyte lysate system. 35 S-methionine-labeled proteins were visualized by autoradiography. ( B and C ) Band intensities from the IVT assay were quantified by densitometric analysis using Imagequant software (Molecular Dynamics) and graphed to demonstrate absolute intensities ( B ) or relative fold increase of EBNA1 encoded by codon-modified GAr domains compared with EBNA1 encoded by native GAr domains ( C ). ( D ) Western blot of EBV-negative HEK293 cells transfected with expression constructs encoding E1-GFP (lane 1), E1ΔGA-GFP (lane 2), E1-GAr(100N)-GFP (lane 3), E1-GAr(100M)-GFP (lane 4), E1-GAr(200N)-GFP (lane 5), E1-GAr(200M)-GFP (lane 6), E1-GAr(300N)-GFP (lane 7), E1-GAr(300M)-GFP (lane 8), E1-GAr(400N)-GFP (lane 9), E1-GAr(400M)-GFP (lane 10), E1-GAr(500N)-GFP (lane 11), or E1-GAr(500M)-GFP (lane 12) with a GFP antibody ( Upper ) or a monoclonal actin antibody ( Lower ). Molecular weight markers M r (kDa) are indicated on the left. ( E ) Band intensities after immunoblotting were quantified as described for B . Representative data from one of four experiments are presented here.

    Article Snippet: EBNA1/pcDNA3 expression constructs were linearized with XbaI and 1 μg of template transcribed with T7 RNA polymerase by using a Riboprobe in vitro transcription system (Promega) supplemented with 50 μCi [α-32 P]UTP (Amersham Biosciences).

    Techniques: Modification, Sequencing, Expressing, Construct, In Vitro, Labeling, Autoradiography, Software, Western Blot, Transfection, Molecular Weight

    YAP1 is activated by platelets and is indispensable for platelet-induced anoikis resistance. a , b Western blot analysis of phosphorylated YAP1 (S127 and S397) and total YAP1 in HEYA8 a and OVCAR8 b cells after two hours under low-attachment conditions with or without platelet co-incubation. GAPDH was used as a loading control ( n = 5). c , d Western blot analysis of phosphorylated YAP1 (S127) and total YAP1 in SKOV3, MDAH-2774, OVCAR4, SW620 c and OVCAR5, OVCA432 and RKO d GAPDH was used as a loading control ( n = 3). e , f Immunofluorescence staining of YAP1 in HEYA8 e and OVCAR8 f cells after two hours under low-attachment conditions with ( lower panels ) or without ( upper panels ) platelet co-incubation. Inlets showing higher magnification of cells on the right side of the panels. Nuclear counterstain was done using Hoechst 33342 ( n = 3). Scale bars = 20 µm. g QRT–PCR and western blot analysis in HEYA8 cells showing efficiency of YAP1 knockdown on the RNA and protein level using two different siRNAs ( n = 3). h Bar graphs showing number of dead (SYTOX Red positive, black ) and living (SYTOX Red negative, red ) HEYA8 cells after 72 h of low attachment and 96 h after siRNA transfection ( n = 3, two-sided Student’s t -test). i QRT–PCR and western blot analysis in OVCAR8 cells showing efficiency of YAP1 knockdown on the RNA and protein level using two different siRNAs ( n = 3). j Bar graphs showing number of dead (SYTOX Red positive, black ) and living (SYTOX Red negative, red ) OVCAR8 cells after 72 h of low attachment and 96 h after siRNA transfection ( n = 3, two-sided Student’s t -test). Bars and error bars represent mean values and the corresponding SEMs (* p

    Journal: Nature Communications

    Article Title: Platelets reduce anoikis and promote metastasis by activating YAP1 signaling

    doi: 10.1038/s41467-017-00411-z

    Figure Lengend Snippet: YAP1 is activated by platelets and is indispensable for platelet-induced anoikis resistance. a , b Western blot analysis of phosphorylated YAP1 (S127 and S397) and total YAP1 in HEYA8 a and OVCAR8 b cells after two hours under low-attachment conditions with or without platelet co-incubation. GAPDH was used as a loading control ( n = 5). c , d Western blot analysis of phosphorylated YAP1 (S127) and total YAP1 in SKOV3, MDAH-2774, OVCAR4, SW620 c and OVCAR5, OVCA432 and RKO d GAPDH was used as a loading control ( n = 3). e , f Immunofluorescence staining of YAP1 in HEYA8 e and OVCAR8 f cells after two hours under low-attachment conditions with ( lower panels ) or without ( upper panels ) platelet co-incubation. Inlets showing higher magnification of cells on the right side of the panels. Nuclear counterstain was done using Hoechst 33342 ( n = 3). Scale bars = 20 µm. g QRT–PCR and western blot analysis in HEYA8 cells showing efficiency of YAP1 knockdown on the RNA and protein level using two different siRNAs ( n = 3). h Bar graphs showing number of dead (SYTOX Red positive, black ) and living (SYTOX Red negative, red ) HEYA8 cells after 72 h of low attachment and 96 h after siRNA transfection ( n = 3, two-sided Student’s t -test). i QRT–PCR and western blot analysis in OVCAR8 cells showing efficiency of YAP1 knockdown on the RNA and protein level using two different siRNAs ( n = 3). j Bar graphs showing number of dead (SYTOX Red positive, black ) and living (SYTOX Red negative, red ) OVCAR8 cells after 72 h of low attachment and 96 h after siRNA transfection ( n = 3, two-sided Student’s t -test). Bars and error bars represent mean values and the corresponding SEMs (* p

    Article Snippet: Quantative reverse transcription –PCR analysis For mRNA quantification, total RNA was isolated using the Direct-zol RNA MiniPrep kit (Zymo Research Corp.) and cDNAs were synthesized using the Verso cDNA kit (Thermo Scientific) per the manufacturer’s instructions.

    Techniques: Western Blot, Incubation, Immunofluorescence, Staining, Quantitative RT-PCR, Transfection