Journal: Virology Journal

Article Title: BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection

doi: 10.1186/s12985-015-0266-8

Figure Lengend Snippet: Curcumin decreases BRCA1 occupancy at the HIV-1 LTR. A . TZM-bl cells were transfected with pcTat and treated the next day with vehicle (DMSO) or a titration of curcumin (0.5, 1, 10, and 20 μM). Samples were analyzed by western blot. Inset depicts a dose–response curve of BRCA1 protein abundance versus curcumin treatment based on the average densitometry counts with error bars representing standard error of three independent measurements. Western blot is representative of three independent experiments. Densitometry counts were taken from three independent treatments to acquire a dose–response curve of BRCA1 expression inhibition (inset plot) B . TZM-bl cells were transfected with pcTat and treated the next day with DMSO or 20 μM curcumin. Bright-Glo luciferase assays and CellTiter-Glo cell viability assays were performed 24 hours post-treatment as described by the manufacturer. Data was normalized to cells containing Tat and treated with DMSO as baseline for Tat-dependent LTR activation. Transfection and treatment assays were performed in triplicate and data plotted represents averaged data of two independent experiments. Error bars show the standard error of two averaged independent measurements. Viability assays were performed in triplicate. C . TZM-bl cells were transfected with pcTat and treated the next day with DMSO or curcumin (20 μM) for 24 hours prior to being collected for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), anti-IgG (10 μg), and anti-RNA polymerase II (RNAP II, 10 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. Single asterisk indicates p

Article Snippet: Quantitative PCR was performed using SYBR Green PCR Master Mix (#4309155, Applied Biosystems, Foster City, CA) with 5 μl of immunoprecipitated material, 0.2 μM of primer [HIV-1 LTR (−69 − +175) Forward 5′-CTGGGCGGGACTGGGGAG-3′ and Reverse 5′-TCACACAACAGACGGGCACAC-3′].

Techniques: Transfection, Titration, Western Blot, Expressing, Inhibition, Luciferase, Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction, Immunoprecipitation

Journal: Virology Journal

Article Title: BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection

doi: 10.1186/s12985-015-0266-8

Figure Lengend Snippet: BRCA1 is present at the HIV-1 LTR in HIV infected T-cells. A . CEM cells were infected with NL4-3 virus (p24 = 5000 pg/ml) for 4 hours and collected 72 hours post-infection for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), anti-V5 (10 μg), and anti-histone H3-phosphorylated at S10 (pS10-H3, 5 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. B . CEM cells were pre-treated with DMSO or 10 μM ATM inhibitor (ATM in ) for 2 hours. Cells were then infected with NL4-3 virus (p24 = 5000 pg/ml) for 4 hours, followed by post-treating the cells with DMSO or ATM in . Cells were collected 72 hours post-infection for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), p-BRCA1 S1423 (10 μg), and anti-V5 (10 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. Double asterisk indicates statistically significant difference p ≤ 0.01.

Article Snippet: Quantitative PCR was performed using SYBR Green PCR Master Mix (#4309155, Applied Biosystems, Foster City, CA) with 5 μl of immunoprecipitated material, 0.2 μM of primer [HIV-1 LTR (−69 − +175) Forward 5′-CTGGGCGGGACTGGGGAG-3′ and Reverse 5′-TCACACAACAGACGGGCACAC-3′].

Techniques: Infection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction, Immunoprecipitation

Journal: Scientific Reports

Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

doi: 10.1038/s41598-020-58586-3

Figure Lengend Snippet: Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).

Article Snippet: Kit extractions We tested three different silica-column kits: Zymo ZR Viral DNA/RNA Kit (outdated protocol, D7021), Zymo Quick-DNA/RNA Kit (updated protocol, D7021), and the QIAquick PCR Purification Kit (28104, Qiagen).

Techniques: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Purification

Journal: BMC Biology

Article Title: Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins

doi: 10.1186/1741-7007-2-21

Figure Lengend Snippet: ChIP analysis of histone modification at the SYBL1 promoter region. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using an XhoI polymorphism in the 5' UTR of the SYBL1 gene in the Xq28 pseudoautosomal region that is also present on the Y chromosome. In normal male cells, the Y-linked locus is inactivated, hypermethylated, and late-replicating as is the inactive X allele in female cells [23,31,49]. To determine if this region has abnormal histone modifications on the X-inactivated but DNA hypomethylated ICF female X, ChIP assays were performed for acetylated histone H3 (acH3) and K4-methylated histone H3 (mK4) in normal male lymphoblasts and in PT3 ICF female fibroblasts; the ethidium bromide-stained gels of each sample are shown before and after digestion with XhoI. The undigested alleles (XhoI+) are 268 bp; the digested alleles (XhoI-) result in fragments of 108 and 260 bp. The ChIP assay for acetylated histone H3 (acH3) shows that only the XhoI-digested allele (XhoI+) is hyperacetylated in a normal male lymphoblast (NMLB1), and this corresponds to the active X allele (Xa) by RT-PCR (data not shown). An hTERT-transformed clone of PT3 ICF fibroblasts was also analyzed by ChIP. This clone has normal monoallelic expression of SYBL1 even though the promoter region is extremely hypomethylated as determined by bisulfite methylation analysis of DNA. The inactive X allele in the PT3 clone is hypoacetylated at histone H3 and hypomethylated at H3K4 because only the active X allele (XhoI-) is immunoprecipitated with either the acetylated or K4-methylated histone H3 antibodies (although a small portion of the inactive X also appears to have been precipitated by the acetylated H3 antibody).

Article Snippet: After elution of immune complexes, they were heated at 65°C for 4 h to reverse crosslinks, and the DNA was recovered with a "QIAquick" PCR purification kit from Qiagen Inc. (Valencia, CA).

Techniques: Chromatin Immunoprecipitation, Modification, Methylation, Staining, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Expressing, Immunoprecipitation

Journal: PLoS Genetics

Article Title: Parent-of-Origin Effects Implicate Epigenetic Regulation of Experimental Autoimmune Encephalomyelitis and Identify Imprinted Dlk1 as a Novel Risk Gene

doi: 10.1371/journal.pgen.1004265

Figure Lengend Snippet: Transgenic overexpression of Dlk1 modulates EAE severity and adaptive immune responses. A) Dlk1 mRNA expression in immune tissues in Dlk1 transgenic and wild type littermate control mice measured using TaqMan PCR. Relative Dlk1 expression was calculated in relation to the mean of housekeeping gene, beta-2 microglobulin, using 2-ΔΔCt method (N = 3–5 mice/group). B) Daily EAE scores in Dlk1 transgenic (N = 24) and wild type littermate control (N = 20) mice after immunization with MOG demonstrate that the higher levels of Dlk1 in transgenic mice are protective against EAE. Mean EAE clinical score (and the standard error of the mean) is given for each day post immunization (p.i.). The figure represents pooled data from three separate experiments. Mann-Whitney U-test was used to compare the daily EAE scores (p-value

Article Snippet: Quantitative real-time PCR of rat Dlk1 , Rtl1 and Dio3 in the BC material was performed using a BioRad CFX384 Touch real-time PCR system with a two-step PCR protocol (95°C for 3 min. followed by 40 cycles of 95°C for 10 sec., 60°C for 30 sec. followed by melt curve analysis), using SYBR Green as the fluorophore (Bio-Rad).

Techniques: Transgenic Assay, Over Expression, Expressing, Mouse Assay, Polymerase Chain Reaction, MANN-WHITNEY, Significance Assay

Journal: Journal of Alzheimer's disease : JAD

Article Title: Increased expression of readthrough acetylcholinesterase variants in the brains of Alzheimer's disease patients

doi: 10.3233/JAD-160220

Figure Lengend Snippet: Transcript levels of the “readthrough” acetylcholinesterase (AChE-R) variant are increased in cerebral cortex of AD subjects Relative mRNA levels of the transcripts for AChE-T (or “tailed”), AChE-R (or “readthrought”) and N-AChE (or N-extended) splice variants and for proline-rich membrane anchor 1 (PRiMA-1) were analysed by q RT-PCR in frontal cortex of NDC (n= 22) and AD subjects (n= 19). For AChE transcript analysis specific primers with Power SYBR® Green PCR Master Mix were employed and the specificity of the PCR products was confirmed by dissociation curve analysis. PRiMA-1 transcripts were measured using a specific TaqMan GenExpression Assay with TaqMan PCR Master Mix. Transcript levels were calculated by the comparative 2 −ΔCt method with respect to GAPDH. The results were confirmed in two independent determinations. Mean value ± SEM are represented. *Significantly increased ( p

Article Snippet: First-strand cDNAs were synthesized by reverse transcription of 1.5 μg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Life Technologies Paisley, UK), according to the manufacturer's instructions. q RT-PCR amplification was performed using StepOne-Plus™ Real-Time PCR System with Power SYBR® Green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions for analysis of AChE transcripts.

Techniques: Variant Assay, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 ?-1,6-N-acetylglusaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas

doi: 10.1186/1471-2407-9-79

Figure Lengend Snippet: C2GnT1 mRNA levels were upregulated in human colorectal adenocarcinomas compared to normal tissues . (A) Graph of amplification plots obtained by the Applied Biosystems 7500 Real-Time PCR system for C2GnT1 mRNA expression. (B) A box-plot showing the results of semi-quantitative RT-PCR analysis. The distribution of mRNA gene expression is shown as a fold change in C2GnT1 mRNA expression in normal colorectal tissues and in well, moderately, and poorly differentiated colorectal adenocarcinomas. A Kruskal-Wallis non-parametric test was performed for statistical comparison among the groups. a Denotes a significant difference (p = 0.015) between normal and well groups; b Denotes a significant difference (p = 0.025) between normal and moderate groups.

Article Snippet: The cDNAs were amplified in an Applied Biosystems 7500 Real-Time PCR system using a reaction volume of 7.5 μl containing 1× SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and 15 pmol/μL of each primer.

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

Journal: British Journal of Pharmacology

Article Title: PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity

doi: 10.1111/j.1476-5381.2010.00945.x

Figure Lengend Snippet: Chemical structure and DNA binding properties of PM01183. (A) Structure of PM01183; (B) binding to naked DNA. Drugs were incubated with a 250 bp PCR product at 25°C during 1 h and the electrophoresis was run in 2% agarose-TAE; (C) relative binding affinities for different DNA triplet sequences. PM01183 was incubated with the labelled double-strand oligodeoxynucleotides at 25°C during 1 h; then the melting assay was started in a 7500 Fast Real-Time PCR System by increasing the temperature up to 95°C in small steps of 1°C·min −1 . Analysis of the 1/C 50 parameter (drug affinity for specific DNA sequences) and ΔT m (max) (stability of the drug–DNA complex) were analysed with an in-house developed Visual Basic Application (VBA) running on Microsoft Excel. The values correspond to the mean and standard deviation from at least two independent experiments for each sequence.

Article Snippet: For the experiments, we followed the methodology previously described ( ; ) using a 7500 Fast Real-Time PCR System (ABI Prism, Applied Biosystems, Foster City, CA, USA).

Techniques: Binding Assay, Incubation, Polymerase Chain Reaction, Electrophoresis, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing

Journal: Scientific Reports

Article Title: Levodopa/benserazide microsphere (LBM) prevents L-dopa induced dyskinesia by inactivation of the DR1/PKA/P-tau pathway in 6-OHDA-lesioned Parkinson's rats

doi: 10.1038/srep07506

Figure Lengend Snippet: mRNA expression of DR1,PKA, PPEB, tau were evaluated by RT-PCR from the ipsilateral sides of the striatum. mRNA were assessed in extracts from sham group, 6-OHDA-lesioned rats treated with vehicle, pulsatile L-dopa (20 mg/kg, bid) or LBM-L (20 mg/kg), LBM-M (40 mg/kg) and LBM-H (60 mg/kg). (A) DR1 mRNA levels expressed relative to GAPDH mRNA; (B) PKA mRNA levels expressed relative to GAPDH mRNA; (C) PPEB mRNA levels expressed relative to GAPDH mRNA; (D) tau mRNA levels expressed relative to GAPDH mRNA; Results are expressed by RQ from the ABI 7500 and normalized using the sham groups; * p

Article Snippet: Calculations of threshold cycle and difference were analyzed with ABI 7500 Real-Time PCR System (Life Technologies, Carlsbad, CA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Oncotarget

Article Title: An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation

doi: 10.18632/oncotarget.7502

Figure Lengend Snippet: Analysis of mRNA and protein levels for pRb and E2F1 during EBV primary infection at 2, 5, and 7 dpi A. Human PBMCs were infected by BACEBV-GFPWT (WT) and EBVGFPΔE3C130-159 (ΔE3C130-159) virus and cells were harvested at 2, 5, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of pRb and E2F1 were quantified by qRT-PCR on a StepOnePlus real-time PCR system. B. The protein levels of pRb and E2F1 in PBMC infected with BACEBV-GFPWT and EBVGFPΔE3C130-159 virus at 2, 5 and 7 days p.i. were analyzed Western blot. dpi, days post-infection; RQ, relative quantity; RI, relative intensity. *P

Article Snippet: RT-qPCR was performed on a StepOnePlus real-time PCR System (Applied Biosystems Inc, Carlsbad, CA).

Techniques: Infection, Synthesized, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncotarget

Article Title: An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation

doi: 10.18632/oncotarget.7502

Figure Lengend Snippet: Analysis of mRNA and protein levels for p53, Mdm2, CyclinD1 and Cdk6 during EBV primary infection at 2, 5, and 7 dpi A. Human PBMCs were infected by BACEBV-GFPWT (WT) and EBVGFPΔE3C130-159 (ΔE3C130-159) virus and cells were harvested at 2, 5, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of p53, Mdm2, CyclinD1 and Cdk6 were quantified by qRT-PCR on a StepOnePlus real-time PCR system. B. The protein levels of p53, Mdm2, CyclinD1 and Cdk6 in PBMC infected with BACEBV-GFPWT and EBVGFPΔE3C130-159 virus at 2, 5 and 7 days p.i. were analyzed Western blot. dpi, days post-infection; RQ, relative quantity; RI, relative intensity. *P

Article Snippet: RT-qPCR was performed on a StepOnePlus real-time PCR System (Applied Biosystems Inc, Carlsbad, CA).

Techniques: Infection, Synthesized, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular and cellular neurosciences

Article Title: Transcriptional Regulation of N-acetylaspartate Metabolism in the 5xFAD Model of Alzheimer's Disease: Evidence for Neuron-Glia Communication During Energetic Crisis

doi: 10.1016/j.mcn.2015.03.009

Figure Lengend Snippet: ( A ) In-house QRT-PCR confirmation of the most significantly downregulated Complex I gene, Ndusf2 , confirms 4-month array data (5xFAD values expressed as fold-2 month wild type values, +/- SEM, n=5. ** p≤0.01). Reduced Ndusf2 expression correlates

Article Snippet: QRT-PCR was performed with Syber Green on an Applied Biosystems Step One Real Time PCR system (Applied Biosystems, Foster City, Ca).

Techniques: Quantitative RT-PCR, Expressing

Journal: Molecular and cellular neurosciences

Article Title: Transcriptional Regulation of N-acetylaspartate Metabolism in the 5xFAD Model of Alzheimer's Disease: Evidence for Neuron-Glia Communication During Energetic Crisis

doi: 10.1016/j.mcn.2015.03.009

Figure Lengend Snippet: ( A ) Increased aspa expression in 5xFAD brains at 2 months of age relative to wild type as assessed by QRT-PCR. Aspa expression presented as fold-wild type 2 month levels for each group, mean +/- SEM, n=5. Immunhistochemical evidence of increases in ASPA

Article Snippet: QRT-PCR was performed with Syber Green on an Applied Biosystems Step One Real Time PCR system (Applied Biosystems, Foster City, Ca).

Techniques: Expressing, Quantitative RT-PCR

Journal: Molecular and cellular neurosciences

Article Title: Transcriptional Regulation of N-acetylaspartate Metabolism in the 5xFAD Model of Alzheimer's Disease: Evidence for Neuron-Glia Communication During Energetic Crisis

doi: 10.1016/j.mcn.2015.03.009

Figure Lengend Snippet: Whole brain homogenates analyzed by HPLC show a significant reduction in NAA from 2-4 months of age in 5xFAD brains ( 1A ; mean +/- SEM shown for each genotype at each age, n=7). QRT-PCR analysis of Nat8L expression 2-4 months of age showing a significant

Article Snippet: QRT-PCR was performed with Syber Green on an Applied Biosystems Step One Real Time PCR system (Applied Biosystems, Foster City, Ca).

Techniques: High Performance Liquid Chromatography, Quantitative RT-PCR, Expressing

Journal: Journal of Bacteriology

Article Title: Temporal Analysis of Coxiella burnetii Morphological Differentiation

doi: 10.1128/JB.186.21.7344-7352.2004

Figure Lengend Snippet: Relative expression levels of selected C. burnetii genes during morphological differentiation as detected by quantitative RT-PCR. Assays were performed using TaqMan primers and probes specific for each gene. Vero cell monolayers were incubated with purified SCV for 1 h to allow for adherence and internalization. Extracellular organisms were then washed from cell monolayers, and fresh medium was added. This time was designated as 0 h p.i. Total RNA was extracted at the indicated times. Transcriptional activity is expressed as relative expression, with transcript copy number normalized to the number of C. burnetii genomes present in each sample. The results are expressed as the mean from three experiments, with error bars representing the standard error of the mean.

Article Snippet: QPCR and reverse transcriptase PCR (RT-PCR) (see below) were performed using TaqMan Universal PCR Master Mix and a Prism 7000 sequence detection system (Applied Biosystems).

Techniques: Expressing, Quantitative RT-PCR, Incubation, Purification, Activity Assay

Journal: PLoS ONE

Article Title: The Epithelial-Mesenchymal Transition (EMT) Regulatory Factor SLUG (SNAI2) Is a Downstream Target of SPARC and AKT in Promoting Melanoma Cell Invasion

doi: 10.1371/journal.pone.0040378

Figure Lengend Snippet: mRNA levels of SPARC and SLUG in melanoma cells at various stages of tumor development. ( A ) Analysis of SPARC and SLUG mRNA levels: relative gene expression levels of SPARC and SLUG in cultures of melanoma cells derived from RGP, VGP or metastatic melanoma tumors were evaluated by relative Q-PCR using an ABI Biosystems 7900HT Fast Real Time PCR System and the SYBR Green dye detection protocol. Data were analyzed using the 2 −ddCt method and human 18S transcript level was used to normalize for each sample. Values are the mean of independent triplicates. RGP, Radial Growth Phase; VGP, Vertical Growth Phase. ( B ) Positive correlation between SPARC and SLUG in melanoma samples: regression analysis to determine the correlation between SPARC and SLUG in human melanoma samples. R , Spearman’s Rank Correlation Coefficient; P

Article Snippet: Quantitative PCR was performed on 25 ng cDNA samples, in sealed 384-well microtiter plates using the SYBR Green™ PCR Master Mix (Applied Biosystems) with the 7900HT Fast Real-Time PCR System (Applied Biosystems).

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

Journal: The Journal of parasitology

Article Title: DIFFERENCES IN CYSTEINE PROTEASE ACTIVITY IN SCHISTOSOMA MANSONI-RESISTANT AND -SUSCEPTIBLE BIOMPHALARIA GLABRATA AND CHARACTERIZATION OF THE HEPATOPANCREAS CATHEPSIN B FULL-LENGTH cDNA

doi: 10.1645/GE-1410R.1

Figure Lengend Snippet: Real-time quantitative RT-PCR analysis of the snail hepatopancreas cathepsin B transcript in resistant (BS-90) and susceptible (NMRI and M-line) pre- and postexposure to miracidia. RT-PCR was performed with the cathepsin B gene-specific primers using RNA from individual snails after various times postexposure. Gene-specific primers for the housekeeping gene myoglobin were used to assess sample uniformity and to confirm that the template cDNA was used in equivalent amounts for each amplification reaction. RT-PCR reactions were performed using an Applied Biosystems 7300 Real-Time PCR System, and detection of the fluorescently labeled product was performed at the end of the amplification period. The -fold increase of gene expression was calculated by comparative Ct method as described in Materials and Methods. A value of > 1-fold increase was considered to be significant (1 = value for nonexposed snails).

Article Snippet: The RT-PCR reactions were performed using an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, California).

Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Labeling, Expressing

Journal: Annals of Laboratory Medicine

Article Title: Performance Evaluation of the Real-Q Cytomegalovirus (CMV) Quantification Kit Using Two Real-Time PCR Systems for Quantifying CMV DNA in Whole Blood

doi: 10.3343/alm.2016.36.6.603

Figure Lengend Snippet: Bland-Altman plot comparing the artus cytomegalovirus (CMV) RG PCR kit and the Real-Q CMV Quantification kit on different real-time PCR platforms: (A) the 7500 Fast Real-time PCR system, (B) the CFX96 real-time PCR detection system. Solid lines are the mean differences between the values; dashed lines are the mean difference plus or minus 1.96 SD (95% confidential interval of mean difference).

Article Snippet: In the present study, we assessed the performance of the Real-Q assay for quantifying CMV DNA in WB using two real-time PCR platforms: the 7500 Fast real-time PCR system (7500 Fast; Applied Biosystems, Foster City, CA, USA) and the CFX96 real-time PCR detection system (CFX96; Bio-Rad, Hercules, CA, USA).

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

Journal: Annals of Laboratory Medicine

Article Title: Performance Evaluation of the Real-Q Cytomegalovirus (CMV) Quantification Kit Using Two Real-Time PCR Systems for Quantifying CMV DNA in Whole Blood

doi: 10.3343/alm.2016.36.6.603

Figure Lengend Snippet: Linearity of the Real-Q cytomegalovirus (CMV) Quantification kit on different real-time PCR platforms: (A) the 7500 Fast real-time PCR system and (B) the CFX96 real-time PCR detection system.

Article Snippet: In the present study, we assessed the performance of the Real-Q assay for quantifying CMV DNA in WB using two real-time PCR platforms: the 7500 Fast real-time PCR system (7500 Fast; Applied Biosystems, Foster City, CA, USA) and the CFX96 real-time PCR detection system (CFX96; Bio-Rad, Hercules, CA, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: Infection and Immunity

Article Title: Vibrio parahaemolyticus Disruption of Epithelial Cell Tight Junctions Occurs Independently of Toxin Production

doi: 10.1128/IAI.73.3.1275-1283.2005

Figure Lengend Snippet: Comparison of PCR and Southern hybridization results for four representative V. parahaemolyticus strains. (A) tdh detection; (B) trh detection; (C) tlh detection. Genes were detected by PCR (left panels) and Southern hybridization (right panels). Genomic

Article Snippet: The tdh and tlh PCR mixtures (final volume, 25 μl) contained 10× PCR buffer (50 mM KCl, 10 mM Tris [pH 8.5], 0.1% gelatin, 1.5% MgCl2 ), each deoxynucleoside triphosphate (Invitrogen Life Technologies, Burlington, Ontario, Canada) at a concentration of 0.25 mM, Taq polymerase (1.25 U; New England Biolabs Ltd., Mississauga, Ontario, Canada), and nuclease-free water (Gibco).

Techniques: Polymerase Chain Reaction, Hybridization

Journal: International Journal of Nanomedicine

Article Title: Zinc oxide nanoparticles selectively induce apoptosis in human cancer cells through reactive oxygen species

doi: 10.2147/IJN.S29129

Figure Lengend Snippet: Quantitative real-time polymerase chain reaction analysis of mRNA levels of apoptotic genes in human lung cancer (HepG2) cells treated with zinc oxide nanoparticles (ZnO NPs) at the concentration of 15 μg/mL for 24 hours. Quantitative real-time polymerase chain reaction was performed by QuantiTect SYBR Green PCR kit using an ABI PRISM 7900HT sequence detection system. The β-actin was used as the internal control to normalize the data. ZnO NP-induced alterations in mRNA levels are expressed in relative quantity compared with those for the respective unexposed control cells. ( A ) p53, ( B ) bax, ( C ) bcl-2, and ( D ) caspase-3. Data represented are mean ± standard deviation of three identical experiments made in triplicate. Note: *Statistically significant difference as compared with the controls ( P

Article Snippet: Quantitative real-time PCR was performed by QuantiTect SYBR Green PCR kit (Qiagen) using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA).

Techniques: Real-time Polymerase Chain Reaction, Concentration Assay, SYBR Green Assay, Polymerase Chain Reaction, Sequencing, Standard Deviation

Journal: BMC Medical Genomics

Article Title: Midkine is a NF-?B-inducible gene that supports prostate cancer cell survival

doi: 10.1186/1755-8794-1-6

Figure Lengend Snippet: Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P

Article Snippet: Analysis of MDK mRNA expression by real-time quantitative RT-PCR Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit (QIAGEN, Valencia, CA) with on-membrane DNase I digestion to avoid genomic DNA contamination. cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Hypoxia Triggers SENP1 Modulation of Kruppel-Like Factor 15 and Transcriptional Regulation of Arginase 2 in Pulmonary Endothelium

doi: 10.1161/ATVBAHA.117.310660

Figure Lengend Snippet: KLF15 is the major Kruppel-Like Factor in HPMEC and important regulator of critical endothelial enzymes- Arginase 2 and eNOS Total RNA from Human Pulmonary Microvascular endothelial cells was converted to cDNA and the relative mRNA expression of KLF15 was determined in quiescent HPMEC using real-time PCR (N=3), B) Cell lysates from HPMEC, human coronary artery endothelial cells (HCAEC), Human Aortic Endothelial Cells (HAEC), Human Aortic Smooth Muscle Cells (HASMC) were subjected to western blotting with KLF15, eNOS and β-tubulin antibodies (N=4). Cell lysates from HPMEC overexpressing HA-KLF15 was used as a positive control, C) Total RNA from Human Pulmonary Microvascular endothelial cells was converted to cDNA and the relative mRNA expression of KLF2, 4 and 15 was determined in quiescent HPMEC using real-time PCR (N=3) *p

Article Snippet: RNA was then reverse transcribed with oligo dT primers to obtain cDNA and quantitative real time PCR (Applied Biosystems) was performed using SYBR Green Supermix mix (Applied Biosystems) whereas semi-quantitative RTPCR was performed using a conventional Biorad PCR machine and the following primer sets: Human Arg2 : Forward: 5′-GGG CCC TGA AGG CTG TAG-3′, Reverse: 5′-AAT GGA GCC ACT GCC ATC-3′.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Positive Control

Journal: PLoS ONE

Article Title: Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays

doi: 10.1371/journal.pone.0172756

Figure Lengend Snippet: Amplification curves obtained by β 0 39 or β + IVSI-110 genotyping assays from circulating DNA extracted from maternal plasma. Real-time PCR with β 0 39 (A) or β + IVSI-110 (B) genotyping assays, containing a normal probe (VIC ® , upper panels) and a mutated probe (FAM ™ , lower panels) were performed for circulating DNA extracted from maternal plasma where the father was a carrier of β 0 39 (A) or β + IVSI-110 (B) thalassemia mutation, respectively. The plots show the ΔRn values as a function of the number of amplification cycles, while the threshold line is drawn in blue. Samples are listed according to increasing gestational ages. In panel (B), #146a and #146b refer to samples collected from the same pregnant woman (number 146) at different gestational ages: 5 weeks and 10 weeks, respectively. The amplification was performed by using the StepOne ™ Real-Time PCR System (Applied Biosystems ® —Thermo Fisher Scientific).

Article Snippet: The reactions were carried out on a StepOne™ Real-Time PCR System (Applied Biosystems® —Thermo Fisher Scientific), by using the StepOne Software (Applied Biosystems® —Thermo Fisher Scientific).

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

Journal: PLoS ONE

Article Title: Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays

doi: 10.1371/journal.pone.0172756

Figure Lengend Snippet: Amplification curves obtained by β + IVSI-6 or β 0 IVSI-1 genotyping assays from circulating DNA extracted from maternal plasma. Real-time PCR with β + IVSI-6 (A) or β 0 IVSI-1 (B) genotyping assays, containing a normal probe (VIC ® , upper panels) and a mutated probe (FAM ™ , lower panels), were performed for circulating DNA extracted from maternal plasma where the father was a carrier of β + IVSI-6 (A) or β 0 IVSI-1 (B) thalassemia mutation, respectively. The plots show the ΔRn values as a function of the number of amplification cycles, while the threshold line is drawn in blue. Samples are listed according to increasing gestational ages. The amplification was performed by using the StepOne ™ Real-Time PCR System (Applied Biosystems ® —Thermo Fisher Scientific).

Article Snippet: The reactions were carried out on a StepOne™ Real-Time PCR System (Applied Biosystems® —Thermo Fisher Scientific), by using the StepOne Software (Applied Biosystems® —Thermo Fisher Scientific).

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

Journal: PLoS ONE

Article Title: A Multiplex Real-Time PCR Assay to Diagnose and Separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae) in the New World

doi: 10.1371/journal.pone.0142912

Figure Lengend Snippet: Decision tree flowchart developed using the interpretation rules for the triplex real-time PCR assay run on a Bio-Rad CFX96 system.

Article Snippet: Development of interpretation rules The interpretation rules developed here are based on the Cq values obtained from the 452 samples tested with the real-time PCR assay on a Bio-Rad CFX96 Touch Real-time PCR Detection System.

Techniques: Real-time Polymerase Chain Reaction

Journal: BMC Genomics

Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

doi: 10.1186/s12864-017-4371-5

Figure Lengend Snippet: DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

Article Snippet: The ChIP DNA Clean & Concentrator™ (Zymo Research; Zy), the Monarch® PCR & DNA Cleanup Kit (New England Biolabs; Ne), the MinElute PCR Purification Kit (Qiagen, Qm), the QIAquick PCR Purification Kit (Qiagen; Qp), the Agencourt AMPure XP kit (Beckman; Ba) and the RNAClean™ XP kit (Beckman; Br), and phenol/chloroform extraction (Invitrogen; PC) performed well with de-crosslinked chromatin.

Techniques: DNA Purification, Chromatin Immunoprecipitation, Purification, Generated, Derivative Assay, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

Journal: Innate Immunity

Article Title: Study of the activation of the PI3K/Akt pathway by the motif of σA and σNS proteins of avian reovirus

doi: 10.1177/1753425919890648

Figure Lengend Snippet: Identification of the recombinant plasmids by PCR. Lane M: 100-bp DNA ladder; Lane 1: σA-M1-pcAGEN; Lane 2: σA-M2-pcAGEN; Lane 3: σA-M3-pcAGEN; Lane 4: σA-M4-pcAGEN; Lane 5: σA-M5-pcAGEN; Lane 6: σA-M6-pcAGEN; Lane 7: σNS-M1-pcAGEN; Lane 8: σNS-M2-pcAGEN; Lane 9: σNS-M3-pcAGEN.

Article Snippet: To amplify the σA-M1 gene (σA gene mutant 1), the PCR protocol consisted of three rounds of PCR amplification.

Techniques: Recombinant, Polymerase Chain Reaction