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    Structured Review

    Thermo Fisher qpcr amplification
    miR99 family members are downregulated in more radioresistant breast and prostate cancer cell lines and are upregulated following DNA damage. (A) Microarray screen of <t>microRNAs</t> induced by radiation. Y-axis: fold upregulated following irradiation in MCF7. X-axis: fold expression in MCF7 (p53+) vs. SK-BR-3 (p53−). Highlighted microRNAs were at least 2 fold overexpressed in MCF7, and upregulated at least 1.4 fold 24 hours following treatment with IR. miR-99a and 100 were both found to meet these criteria. (B) Clonogenic assay of MCF7 and SK-BR-3 cells following IR. The colonies/well were counted to get fractional survival. Mean + SD (n=3). (C) miR-99a and miR-100 expression measured by <t>qPCR</t> in MCF7 and SK-BR-3 cells, normalized to u6snRNA. Mean + SD (n=6). (D) miR-99a and miR-100 expression measured by qPCR, normalized to β -Actin, 24 hours following IR treatment in MCF7 cells. Mean + SD (n=6). (E) Clonogenic assay of LNCap and C4-2 cells following IR. Fractional survival as in Fig. 1b (n=3). (F) miR-99a and miR-100 expression measured by qPCR in LNCaP and C4-2 cells, normalized to u6snRNA. Mean + SD (n=6). (G) miR-99a and miR-100 expression measured by qPCR, normalized to β -Actin, 24 hours following IR treatment in LNCaP cells. Mean + SD (n=6).
    Qpcr Amplification, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 257 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The miR-99 family regulates the DNA damage response through its target SNF2H"

    Article Title: The miR-99 family regulates the DNA damage response through its target SNF2H

    Journal: Oncogene

    doi: 10.1038/onc.2012.131

    miR99 family members are downregulated in more radioresistant breast and prostate cancer cell lines and are upregulated following DNA damage. (A) Microarray screen of microRNAs induced by radiation. Y-axis: fold upregulated following irradiation in MCF7. X-axis: fold expression in MCF7 (p53+) vs. SK-BR-3 (p53−). Highlighted microRNAs were at least 2 fold overexpressed in MCF7, and upregulated at least 1.4 fold 24 hours following treatment with IR. miR-99a and 100 were both found to meet these criteria. (B) Clonogenic assay of MCF7 and SK-BR-3 cells following IR. The colonies/well were counted to get fractional survival. Mean + SD (n=3). (C) miR-99a and miR-100 expression measured by qPCR in MCF7 and SK-BR-3 cells, normalized to u6snRNA. Mean + SD (n=6). (D) miR-99a and miR-100 expression measured by qPCR, normalized to β -Actin, 24 hours following IR treatment in MCF7 cells. Mean + SD (n=6). (E) Clonogenic assay of LNCap and C4-2 cells following IR. Fractional survival as in Fig. 1b (n=3). (F) miR-99a and miR-100 expression measured by qPCR in LNCaP and C4-2 cells, normalized to u6snRNA. Mean + SD (n=6). (G) miR-99a and miR-100 expression measured by qPCR, normalized to β -Actin, 24 hours following IR treatment in LNCaP cells. Mean + SD (n=6).
    Figure Legend Snippet: miR99 family members are downregulated in more radioresistant breast and prostate cancer cell lines and are upregulated following DNA damage. (A) Microarray screen of microRNAs induced by radiation. Y-axis: fold upregulated following irradiation in MCF7. X-axis: fold expression in MCF7 (p53+) vs. SK-BR-3 (p53−). Highlighted microRNAs were at least 2 fold overexpressed in MCF7, and upregulated at least 1.4 fold 24 hours following treatment with IR. miR-99a and 100 were both found to meet these criteria. (B) Clonogenic assay of MCF7 and SK-BR-3 cells following IR. The colonies/well were counted to get fractional survival. Mean + SD (n=3). (C) miR-99a and miR-100 expression measured by qPCR in MCF7 and SK-BR-3 cells, normalized to u6snRNA. Mean + SD (n=6). (D) miR-99a and miR-100 expression measured by qPCR, normalized to β -Actin, 24 hours following IR treatment in MCF7 cells. Mean + SD (n=6). (E) Clonogenic assay of LNCap and C4-2 cells following IR. Fractional survival as in Fig. 1b (n=3). (F) miR-99a and miR-100 expression measured by qPCR in LNCaP and C4-2 cells, normalized to u6snRNA. Mean + SD (n=6). (G) miR-99a and miR-100 expression measured by qPCR, normalized to β -Actin, 24 hours following IR treatment in LNCaP cells. Mean + SD (n=6).

    Techniques Used: Microarray, Irradiation, Expressing, Clonogenic Assay, Real-time Polymerase Chain Reaction

    2) Product Images from "PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein"

    Article Title: PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein

    Journal: Oncogene

    doi: 10.1038/onc.2011.190

    PAX8 is required for RB protein stability. The effect of PAX8 knockdown on RB expression at ( a ) the transcript and ( b ) the protein levels. ( a ) Total RNA was isolated from the indicated sample at 48 h post-transfection, and subjected to qPCR analysis. Gene expression levels were normalized to the expression of the housekeeping genes, PPIB and YWHAZ , and presented relative to the siControl-transfected samples. The data are means±s.d. of three independent experiments. ( b ) Whole-cell lysates were extracted from siRNA-transfected A498, 786-O, IGROV-1 and K1 cells at 96 h post transfection. The lysates were subjected to immunoblotting using the indicated antibodies. ( c ) Effects of proteasome inhibition on RB depletion in response to PAX8 knockdown. K1 cells were transfected with the indicated siRNA. After 24 h, cells were transfected either with a vector control (−) or with pCMV-RB (+). At 48 h post DNA transfection, the cells were incubated in medium with or without MG132 for 12 h. Whole-cell lysates were then extracted and subjected to immunoblotting using the indicated antibodies. qPCR, quantitative PCR; RB, retinoblastoma; siRNA, small interfering RNA.
    Figure Legend Snippet: PAX8 is required for RB protein stability. The effect of PAX8 knockdown on RB expression at ( a ) the transcript and ( b ) the protein levels. ( a ) Total RNA was isolated from the indicated sample at 48 h post-transfection, and subjected to qPCR analysis. Gene expression levels were normalized to the expression of the housekeeping genes, PPIB and YWHAZ , and presented relative to the siControl-transfected samples. The data are means±s.d. of three independent experiments. ( b ) Whole-cell lysates were extracted from siRNA-transfected A498, 786-O, IGROV-1 and K1 cells at 96 h post transfection. The lysates were subjected to immunoblotting using the indicated antibodies. ( c ) Effects of proteasome inhibition on RB depletion in response to PAX8 knockdown. K1 cells were transfected with the indicated siRNA. After 24 h, cells were transfected either with a vector control (−) or with pCMV-RB (+). At 48 h post DNA transfection, the cells were incubated in medium with or without MG132 for 12 h. Whole-cell lysates were then extracted and subjected to immunoblotting using the indicated antibodies. qPCR, quantitative PCR; RB, retinoblastoma; siRNA, small interfering RNA.

    Techniques Used: Expressing, Isolation, Transfection, Real-time Polymerase Chain Reaction, Inhibition, Plasmid Preparation, Incubation, Small Interfering RNA

    3) Product Images from "Insect Neuropeptide Bursicon Homodimers Induce Innate Immune and Stress Genes during Molting by Activating the NF-?B Transcription Factor Relish"

    Article Title: Insect Neuropeptide Bursicon Homodimers Induce Innate Immune and Stress Genes during Molting by Activating the NF-?B Transcription Factor Relish

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034510

    Burs α−α and burs β−β treatments induced expression of AMP genes and suppression of bacterial growth in adults. Separate groups of adult flies were neck-ligated immediately after emergence and at 1 h post-ligation injected with 60 ng/0.5 µl of r-burs α−β, burs α−α, or burs β−β. The control flies were injected with the purified cell culture transfected with blank pcDNA 3.1 vector. After the indicated incubation periods, expression of the AMP genes was determined by qPCR. (A) AMP transcript levels in wild type adults. For bacterial inhibition assay (B), neck-ligated wild type flies were injected with r-burs α−β heterodimer, burs α−α homodimer, burs β−β homodimer or blank vector transfected sample, respectively, homogenized and centrifuged for 15 min at 16,000 g. The resulting supernatants were challenged with indicated titers of E. coli for 6 h before plating for colony count. The histograms show the means ± SEM, n = 3 biologically independent experiments.
    Figure Legend Snippet: Burs α−α and burs β−β treatments induced expression of AMP genes and suppression of bacterial growth in adults. Separate groups of adult flies were neck-ligated immediately after emergence and at 1 h post-ligation injected with 60 ng/0.5 µl of r-burs α−β, burs α−α, or burs β−β. The control flies were injected with the purified cell culture transfected with blank pcDNA 3.1 vector. After the indicated incubation periods, expression of the AMP genes was determined by qPCR. (A) AMP transcript levels in wild type adults. For bacterial inhibition assay (B), neck-ligated wild type flies were injected with r-burs α−β heterodimer, burs α−α homodimer, burs β−β homodimer or blank vector transfected sample, respectively, homogenized and centrifuged for 15 min at 16,000 g. The resulting supernatants were challenged with indicated titers of E. coli for 6 h before plating for colony count. The histograms show the means ± SEM, n = 3 biologically independent experiments.

    Techniques Used: Expressing, Ligation, Injection, Purification, Cell Culture, Transfection, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Inhibition

    Transcription expression profiles of burs α and burs β subunits and AMP genes in pharate and newly-emerged adults. qPCR analysis (A) of bursicon α and β gene expression in pharate (PA) and eclosed adults (A) 0–24 h after eclosion; qPCR analysis (B) of AMP gene expression in pharate and eclosed adults 0–24 h after eclosion; Transcription expression ( C ) of AMP genes in 24 h-old adults injected with blank vector transfected sample, r-burs α−α homodimer, r-burs β−β homodimer and r-burs α−β heterodimer, respectively.
    Figure Legend Snippet: Transcription expression profiles of burs α and burs β subunits and AMP genes in pharate and newly-emerged adults. qPCR analysis (A) of bursicon α and β gene expression in pharate (PA) and eclosed adults (A) 0–24 h after eclosion; qPCR analysis (B) of AMP gene expression in pharate and eclosed adults 0–24 h after eclosion; Transcription expression ( C ) of AMP genes in 24 h-old adults injected with blank vector transfected sample, r-burs α−α homodimer, r-burs β−β homodimer and r-burs α−β heterodimer, respectively.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Injection, Plasmid Preparation, Transfection

    AMP transcript levels in rk 4 mutant. Newly emerged adult flies were neck-ligated immediately after emergence and at 1 h post-ligation injected with r-burs α−β heterodimer, burs α−α homodimer, burs β−β homodimer or blank vector transfected sample, as described in Figure 3 . RNA was extracted for qPCR analysis of 11 representative genes (A). Larva FB from the mutant was also used to assay the effect of mutation on burs α−α and burs β−β homodimer induced AMP expression (B). The histograms show the means ± SEM, n = 3 biologically independent experiments.
    Figure Legend Snippet: AMP transcript levels in rk 4 mutant. Newly emerged adult flies were neck-ligated immediately after emergence and at 1 h post-ligation injected with r-burs α−β heterodimer, burs α−α homodimer, burs β−β homodimer or blank vector transfected sample, as described in Figure 3 . RNA was extracted for qPCR analysis of 11 representative genes (A). Larva FB from the mutant was also used to assay the effect of mutation on burs α−α and burs β−β homodimer induced AMP expression (B). The histograms show the means ± SEM, n = 3 biologically independent experiments.

    Techniques Used: Mutagenesis, Ligation, Injection, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Expressing

    Burs α−α and burs β−β treatments induced expression of AMP genes and suppression of bacterial growth in larva FB. FB was dissected from early wondering 3 rd instar larvae and incubated with r-burs α−α or burs β−β homodimer or r-burs α+β heterodimer for 0.5, 1 and 3 h. The control group received the blank vector transfected sample. After incubation, RNA was extracted for qPCR analysis of 4 representative AMP genes (A). For bacterial inhibition assay, FB was homogenized and centrifuged at 16000 g for 20 min at 4°C. The resulting supernatants were challenged with indicated titers of E. coli for 6 h before plating for colony count (B). The histograms show the means ± SEM, n = 3 biologically independent experiments.
    Figure Legend Snippet: Burs α−α and burs β−β treatments induced expression of AMP genes and suppression of bacterial growth in larva FB. FB was dissected from early wondering 3 rd instar larvae and incubated with r-burs α−α or burs β−β homodimer or r-burs α+β heterodimer for 0.5, 1 and 3 h. The control group received the blank vector transfected sample. After incubation, RNA was extracted for qPCR analysis of 4 representative AMP genes (A). For bacterial inhibition assay, FB was homogenized and centrifuged at 16000 g for 20 min at 4°C. The resulting supernatants were challenged with indicated titers of E. coli for 6 h before plating for colony count (B). The histograms show the means ± SEM, n = 3 biologically independent experiments.

    Techniques Used: Expressing, Incubation, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Inhibition

    4) Product Images from "G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV"

    Article Title: G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw038

    ( A ) PhenDC3 treatment accumulated BCBL-1 cells in late S and G2 phase of the cell cycle. Cell cycle profile of PI stained BCBL-1 cells, untreated (black), DMSO treated (black), 10 μM PhenDC3 treated (blue) and 20 μM PhenDC3 treated (red), for 24 h. ( B and C ) BCBL-1 cells treated with 20 μM PhenDC3 show reduced cell proliferation. CFSE-labeled BCBL-1 cells treated with ( B ) DMSO and ( C ) 20 μM PhenDC3, for a period of 72 h. ( D ) BCBL-1 cells treated with 20 μM PhenDC3 show reduction in KSHV genome copies: Total genomic DNA from BCBL-1 cells treated with DMSO (blue) or 20 μM PhenDC3 (red), analyzed for KSHV genome copies by real-time qPCR using KSHV ORF73 primers normalized to respective GAPDH showed reduction. Relative reduction of GAPDH was also calculated for up to 4 days of PhenDC3 treatment. ( E ) MTT assay on BCBL-1 cells treated with 20 μM PhenDC3 showed a moderate decrease in metabolic rate. Analysis of cellular proliferation on BCBL-1 cells treated with DMSO (blue bar) or 20 μM PhenDC3 (red bar), for a period of 4 days.
    Figure Legend Snippet: ( A ) PhenDC3 treatment accumulated BCBL-1 cells in late S and G2 phase of the cell cycle. Cell cycle profile of PI stained BCBL-1 cells, untreated (black), DMSO treated (black), 10 μM PhenDC3 treated (blue) and 20 μM PhenDC3 treated (red), for 24 h. ( B and C ) BCBL-1 cells treated with 20 μM PhenDC3 show reduced cell proliferation. CFSE-labeled BCBL-1 cells treated with ( B ) DMSO and ( C ) 20 μM PhenDC3, for a period of 72 h. ( D ) BCBL-1 cells treated with 20 μM PhenDC3 show reduction in KSHV genome copies: Total genomic DNA from BCBL-1 cells treated with DMSO (blue) or 20 μM PhenDC3 (red), analyzed for KSHV genome copies by real-time qPCR using KSHV ORF73 primers normalized to respective GAPDH showed reduction. Relative reduction of GAPDH was also calculated for up to 4 days of PhenDC3 treatment. ( E ) MTT assay on BCBL-1 cells treated with 20 μM PhenDC3 showed a moderate decrease in metabolic rate. Analysis of cellular proliferation on BCBL-1 cells treated with DMSO (blue bar) or 20 μM PhenDC3 (red bar), for a period of 4 days.

    Techniques Used: Staining, Labeling, Real-time Polymerase Chain Reaction, MTT Assay

    5) Product Images from "Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus"

    Article Title: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus

    Journal: Viruses

    doi: 10.3390/v11111039

    Early impact of different classes of HCV inhibitors on HCV RNA and NS5A. ( A ) Huh-7.5.1 cells were infected with HCV Jc1/Gluc2A at an MOI of 0.5. After 48 h, the cells were treated with various inhibitors (Danoprevir, 0.32 μM; Ledipasvir, 3 μM; Daclatasvir, 3.2 nM; Sofosbuvir, 20 μM), then after a further 8 h cells were fixed and probed sequentially for (−)RNA, (+)RNA and NS5A. Finally, nuclei were stained with DAPI. Cells were imaged on a Leica SP8 confocal microscope using a 63× oil-immersion objective. ( B ) Representative images from each inhibitor showing (−)RNA in green, (+)RNA in red, NS5A in gray and nuclei in blue. Boxed regions shown enlarged. Scale bars represent 10 µm. Early impact of different classes of HCV inhibitors on HCV RNA and NS5A. (C and D) Fields of view were captured as described in Figure 3 , then analyzed using Gen5 software (BioTek). Abundance of (+) and (−)RNA strands was quantified by: ( C ) number of RNA foci per cell; ( D ) RNA fluorescence intensity per cell in the field of view; and ( E ) RT-qPCR to determine copy numbers of the of (+) and (−)RNA using specific primers. All values are expressed as a percentage of the DMSO-treated sample. Error bars indicate standard error of the mean for three independent experiments. Significance of differences between DMSO and drug treatments was calculated using one-way ANOVA and Tukey’s post-test for multiple comparisons. Differences were not significant unless otherwise indicated. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. P values shown on columns indicate comparison to DMSO. Lines indicate comparisons between test samples. Lines group samples, and are color coordinated for (+)RNA and (−)RNA. Flat lines group all samples beneath them, inverted Vs indicate comparison of the samples under the ends of the lines. DNV, Danoprevir; LDV, Ledipasvir; DCV, Daclatasvir; SOF, Sofosbuvir. ( F ) Comparison of intracellular HCV transcript number under control (DMSO) conditions as determined by strand-specific RT-qPCR and bDNA FISH. Error bars indicate standard error of the mean; no additional statistical analyses were performed.
    Figure Legend Snippet: Early impact of different classes of HCV inhibitors on HCV RNA and NS5A. ( A ) Huh-7.5.1 cells were infected with HCV Jc1/Gluc2A at an MOI of 0.5. After 48 h, the cells were treated with various inhibitors (Danoprevir, 0.32 μM; Ledipasvir, 3 μM; Daclatasvir, 3.2 nM; Sofosbuvir, 20 μM), then after a further 8 h cells were fixed and probed sequentially for (−)RNA, (+)RNA and NS5A. Finally, nuclei were stained with DAPI. Cells were imaged on a Leica SP8 confocal microscope using a 63× oil-immersion objective. ( B ) Representative images from each inhibitor showing (−)RNA in green, (+)RNA in red, NS5A in gray and nuclei in blue. Boxed regions shown enlarged. Scale bars represent 10 µm. Early impact of different classes of HCV inhibitors on HCV RNA and NS5A. (C and D) Fields of view were captured as described in Figure 3 , then analyzed using Gen5 software (BioTek). Abundance of (+) and (−)RNA strands was quantified by: ( C ) number of RNA foci per cell; ( D ) RNA fluorescence intensity per cell in the field of view; and ( E ) RT-qPCR to determine copy numbers of the of (+) and (−)RNA using specific primers. All values are expressed as a percentage of the DMSO-treated sample. Error bars indicate standard error of the mean for three independent experiments. Significance of differences between DMSO and drug treatments was calculated using one-way ANOVA and Tukey’s post-test for multiple comparisons. Differences were not significant unless otherwise indicated. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. P values shown on columns indicate comparison to DMSO. Lines indicate comparisons between test samples. Lines group samples, and are color coordinated for (+)RNA and (−)RNA. Flat lines group all samples beneath them, inverted Vs indicate comparison of the samples under the ends of the lines. DNV, Danoprevir; LDV, Ledipasvir; DCV, Daclatasvir; SOF, Sofosbuvir. ( F ) Comparison of intracellular HCV transcript number under control (DMSO) conditions as determined by strand-specific RT-qPCR and bDNA FISH. Error bars indicate standard error of the mean; no additional statistical analyses were performed.

    Techniques Used: Infection, Staining, Microscopy, Software, Fluorescence, Quantitative RT-PCR, Fluorescence In Situ Hybridization

    6) Product Images from "Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells"

    Article Title: Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells

    Journal: Oncology Reports

    doi: 10.3892/or.2018.6329

    Induction of apoptosis in A375 cells by Licochalcone D (LD) treatment. (A) Cell morphological changes were observed by phase-contrast microscopy (magnification, ×200) after treatment with LD (0, 30, 60 and 90 µmol/l) for 24 h. (B) Apoptosis was visualized by the appropriate changes in nuclei stained with Hoechst 33258 (blue) (magnification, ×200). (C) The effects of LD on the induction of A375 cell apoptosis were analyzed by FCM analysis. (D) The apoptosis rate as statistically analyzed. (E) RT-PCR analyses of A375 cells to evaluate mRNA expression of Bcl-2, Bax, caspase-3 and caspase-9. (F) qPCR analyses of A375 cells to evaluate mRNA expression of Bcl-2, Bax, caspase-3 and caspase-9, and relative intensities were normalized by levels of GAPDH. The untreated group level was considered as ‘1.0’. Data are presented as means ± SD of at least three independent experiments. *P
    Figure Legend Snippet: Induction of apoptosis in A375 cells by Licochalcone D (LD) treatment. (A) Cell morphological changes were observed by phase-contrast microscopy (magnification, ×200) after treatment with LD (0, 30, 60 and 90 µmol/l) for 24 h. (B) Apoptosis was visualized by the appropriate changes in nuclei stained with Hoechst 33258 (blue) (magnification, ×200). (C) The effects of LD on the induction of A375 cell apoptosis were analyzed by FCM analysis. (D) The apoptosis rate as statistically analyzed. (E) RT-PCR analyses of A375 cells to evaluate mRNA expression of Bcl-2, Bax, caspase-3 and caspase-9. (F) qPCR analyses of A375 cells to evaluate mRNA expression of Bcl-2, Bax, caspase-3 and caspase-9, and relative intensities were normalized by levels of GAPDH. The untreated group level was considered as ‘1.0’. Data are presented as means ± SD of at least three independent experiments. *P

    Techniques Used: Microscopy, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

    7) Product Images from "EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment"

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007061

    Both EcoHIV and MLV infect mouse brain but only EcoHIV-infected mice develop cognitive impairment. A. Design of the experiment. B. Groups of mice were gavaged with cART or vehicle before and during infection with indicated viruses or vehicle. At day 21 after infection, mice were tested for errors (left panel) and latency (middle panel) in finding the hidden platform, the right panel shows latency to reach the visible platform. RT represents the retention trial. C. Two days after completion of RAWM, the same mice were used to test contextual response to fear conditioning training. The percentage of freezing time was calculated for contextual fear memory deficit in each group of mice. D. Mice were euthanized on day 40 after infection and levels of total vDNA in spleen and brain measured by QPCR (left panel) and integrated viral DNA measured by nested QPCR in PM (right panel). All data are mean ± standard errors. * P
    Figure Legend Snippet: Both EcoHIV and MLV infect mouse brain but only EcoHIV-infected mice develop cognitive impairment. A. Design of the experiment. B. Groups of mice were gavaged with cART or vehicle before and during infection with indicated viruses or vehicle. At day 21 after infection, mice were tested for errors (left panel) and latency (middle panel) in finding the hidden platform, the right panel shows latency to reach the visible platform. RT represents the retention trial. C. Two days after completion of RAWM, the same mice were used to test contextual response to fear conditioning training. The percentage of freezing time was calculated for contextual fear memory deficit in each group of mice. D. Mice were euthanized on day 40 after infection and levels of total vDNA in spleen and brain measured by QPCR (left panel) and integrated viral DNA measured by nested QPCR in PM (right panel). All data are mean ± standard errors. * P

    Techniques Used: Infection, Mouse Assay, Real-time Polymerase Chain Reaction

    EcoHIV establishes latent reservoirs in resting CD4 + cells that are inducible by epigenetic modulators. A. CD4 + T cells isolated from spleen at day 25 post-infection were cocultured with uninfected cells in the presence or absence of ABC as indicated prior to vDNA amplification, each symbol represents culture from a single mouse. B. Six weeks after EcoHIV infection, CD4 + splenic T cells were harvested and purified from donor male 129X1/SvJ mice and injected into the recipient female 129X1 nude mice; their PM were collected after one week. Y chromosome and EcoHIV gag RNA levels in PM were determined by QPCR. Each symbol represents a single mouse. C.-D. Twelve weeks after infection, resting CD4 + T cells were purified from the spleen and C. The levels of integrated EcoHIV DNA and D. Genomic RNA were measured by QPCR. E. Eight weeks after EcoHIV infection of mice, resting CD4 + T cells were isolated and exposed to the agents indicated in culture for 2 days prior to collection and measurement of EcoHIV mRNA QPCR. F. Six weeks after infection by EcoHIV-Luc, mice were treated as shown, euthanized and splenic CD4 + cells isolated for measurement of virus RNA expression (left panel) or protein expression (right panel). * P
    Figure Legend Snippet: EcoHIV establishes latent reservoirs in resting CD4 + cells that are inducible by epigenetic modulators. A. CD4 + T cells isolated from spleen at day 25 post-infection were cocultured with uninfected cells in the presence or absence of ABC as indicated prior to vDNA amplification, each symbol represents culture from a single mouse. B. Six weeks after EcoHIV infection, CD4 + splenic T cells were harvested and purified from donor male 129X1/SvJ mice and injected into the recipient female 129X1 nude mice; their PM were collected after one week. Y chromosome and EcoHIV gag RNA levels in PM were determined by QPCR. Each symbol represents a single mouse. C.-D. Twelve weeks after infection, resting CD4 + T cells were purified from the spleen and C. The levels of integrated EcoHIV DNA and D. Genomic RNA were measured by QPCR. E. Eight weeks after EcoHIV infection of mice, resting CD4 + T cells were isolated and exposed to the agents indicated in culture for 2 days prior to collection and measurement of EcoHIV mRNA QPCR. F. Six weeks after infection by EcoHIV-Luc, mice were treated as shown, euthanized and splenic CD4 + cells isolated for measurement of virus RNA expression (left panel) or protein expression (right panel). * P

    Techniques Used: Isolation, Infection, Amplification, Purification, Mouse Assay, Injection, Real-time Polymerase Chain Reaction, RNA Expression, Expressing

    Mouse macrophages are susceptible to EcoHIV but not MLV. A. Expression of CAT-1 was determined in peritoneal cells (PC) and thymus (TH) of C57BL/6 mice by QPCR. B. Ten days after EcoHIV or MLV infection of mice, the indicated tissues were harvested for measurement of viral nucleic acids by QPCR. C. Eight weeks after mouse infection by EcoHIV or MLV, F4/80 + macrophages were purified from PC and integrated viral DNA in unfractionated or F4/80 + PC was measured by nested-QPCR. D. C57BL/6 euthymic and athymic mice were infected by EcoHIV and PC harvested from groups at the times indicated to measure spliced EcoHIV RNA. Mean and standard errors are shown (* P
    Figure Legend Snippet: Mouse macrophages are susceptible to EcoHIV but not MLV. A. Expression of CAT-1 was determined in peritoneal cells (PC) and thymus (TH) of C57BL/6 mice by QPCR. B. Ten days after EcoHIV or MLV infection of mice, the indicated tissues were harvested for measurement of viral nucleic acids by QPCR. C. Eight weeks after mouse infection by EcoHIV or MLV, F4/80 + macrophages were purified from PC and integrated viral DNA in unfractionated or F4/80 + PC was measured by nested-QPCR. D. C57BL/6 euthymic and athymic mice were infected by EcoHIV and PC harvested from groups at the times indicated to measure spliced EcoHIV RNA. Mean and standard errors are shown (* P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Infection, Purification

    EcoHIV infects mice and induces antiviral immune responses but not immunodeficiency in mice. A.-B. Kinetics of production of total EcoHIV DNA (left panels), integrated EcoHIV DNA (middle panels), and genomic EcoHIV RNA (right panels) were measured by QPCR in A. spleen and B. PC at the times indicated after infection. Each point represents a single mouse. C. EcoHIV gag RNA burden at each time point in copies per ml of whole blood was measured at the times after infection indicated. The dashed line indicates limit of detection of 10 copies. D. The frequency of interferon-γ expression by CD4 + or CD8 + spleen cells at the time indicated after EcoHIV or mock infection was measured by flow cytometry. E. The number of CD4 + and CD8 + T cells was measured by flow cytometry at the indicated times and the CD4 + :CD8 + ratio is shown. Symbols represent individual mice, the horizontal lines represent the mean. F. Longitudinal anti-NDK Gag antibodies in 3 EcoHIV-infected mice were measured by ELISA at the times indicated after infection. Each panel represents titers in a single mouse, mean +/- standard errors are shown.
    Figure Legend Snippet: EcoHIV infects mice and induces antiviral immune responses but not immunodeficiency in mice. A.-B. Kinetics of production of total EcoHIV DNA (left panels), integrated EcoHIV DNA (middle panels), and genomic EcoHIV RNA (right panels) were measured by QPCR in A. spleen and B. PC at the times indicated after infection. Each point represents a single mouse. C. EcoHIV gag RNA burden at each time point in copies per ml of whole blood was measured at the times after infection indicated. The dashed line indicates limit of detection of 10 copies. D. The frequency of interferon-γ expression by CD4 + or CD8 + spleen cells at the time indicated after EcoHIV or mock infection was measured by flow cytometry. E. The number of CD4 + and CD8 + T cells was measured by flow cytometry at the indicated times and the CD4 + :CD8 + ratio is shown. Symbols represent individual mice, the horizontal lines represent the mean. F. Longitudinal anti-NDK Gag antibodies in 3 EcoHIV-infected mice were measured by ELISA at the times indicated after infection. Each panel represents titers in a single mouse, mean +/- standard errors are shown.

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Infection, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    EcoHIV integrates efficiently in the murine host genome. A. Schematic representation of strategy for assay of integration. In the first round PCR, primers, B1-F and B1-R, from the host B1 repetitive elements and a HIV-specific primer, Fnef, were used to amplify a region of integrated virus and the host flanking region. The second nested QPCR based on the HIV RU5 region was performed using the preamplified products. B. Integration standard curve was generated by logarithmic regression of the amplified IS sample signals. C.-F. Mice (n = 4/system) were pretreated with ABC or RAL 24 h before EcoHIV infection, euthanized 3 days later, and spleens collected for measurement of C. total, D. integrated, and E. 2-LTR circle vDNA; F. spliced vRNA by QPCR of DNA or cDNA as described in Methods.
    Figure Legend Snippet: EcoHIV integrates efficiently in the murine host genome. A. Schematic representation of strategy for assay of integration. In the first round PCR, primers, B1-F and B1-R, from the host B1 repetitive elements and a HIV-specific primer, Fnef, were used to amplify a region of integrated virus and the host flanking region. The second nested QPCR based on the HIV RU5 region was performed using the preamplified products. B. Integration standard curve was generated by logarithmic regression of the amplified IS sample signals. C.-F. Mice (n = 4/system) were pretreated with ABC or RAL 24 h before EcoHIV infection, euthanized 3 days later, and spleens collected for measurement of C. total, D. integrated, and E. 2-LTR circle vDNA; F. spliced vRNA by QPCR of DNA or cDNA as described in Methods.

    Techniques Used: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Generated, Amplification, Mouse Assay, Infection

    8) Product Images from "Roles of the Gac-Rsm pathway in the regulation of phenazine biosynthesis in Pseudomonas chlororaphis 30-84"

    Article Title: Roles of the Gac-Rsm pathway in the regulation of phenazine biosynthesis in Pseudomonas chlororaphis 30-84

    Journal: MicrobiologyOpen

    doi: 10.1002/mbo3.90

    Impact of rsmZ on phenazine and AHL production. (A) Phenazine production by different derivatives in vitro. Treatments included strains with plasmid pLAFR3 containing either no insert (NI) or P tac - rsmZ . Growth conditions were as in ( Fig. 5 A). Phenazine was extracted in benzene and the amount of phenazine was measured at OD 367 . Data points represent means of three replicates ± standard deviations. Similar results were obtained in at least three independent experiments. (B) Promoter activity of the rsmZ gene in WT, gacS , and gacA was determined by β-galactosidase activities. Treatments included strains with plasmid pKT-2 containing either a promoter-less lacZ insertion or the rsmZ promoter fused to the promoter-less lacZ gene (P rsmZ -lacZ ). Insertions were made in front of the promoter-less gfp gene, already present on the plasmid. Bacterial strains were grown in LB medium for 24 h at 28°C with shaking to an OD 600 of ∼1.8. Each value is the average from two different cultures ± standard deviations with two independent experiments. The experiment was repeated at least twice and similar results were obtained. (C) Effect of rsmZ on AHL production. Total AHL extractions were prepared from cell-free supernatants of WT, Δ gacS , or Δ gacA with plasmid pLAFR3 containing either NI or P tac - rsmZ . Cell-free supernatants were made from overnight cultures grown in LB medium (OD 600 ∼1.8). The relative amount of AHL in each extract was determined by β-galactosidase assays using the AHL-specific reporter 30-84I/Z ( phzI − , phzB::lacZ ). Bars represent β-galactosidase activity measured in Miller Units of 30-84I/Z grown with the AHL extracts indicated. Each value is the average from three different cultures ± standard deviations. (D) Relative expression of phzI , phzR , and phzY genes in the gacA mutant (harboring the pLAFR3-P tac - rsmZ ) compared with WT (harboring pLAFR3 empty vector) as determined by qPCR. rpoD was used as the reference gene. Cells were grown in AB minimal medium + 2% casamino acid for 18 h with shaking to an OD 600 of 1.2. Data points represent means of three replicates ± standard deviations. These experiments were repeated at least three times and similar results were obtained.
    Figure Legend Snippet: Impact of rsmZ on phenazine and AHL production. (A) Phenazine production by different derivatives in vitro. Treatments included strains with plasmid pLAFR3 containing either no insert (NI) or P tac - rsmZ . Growth conditions were as in ( Fig. 5 A). Phenazine was extracted in benzene and the amount of phenazine was measured at OD 367 . Data points represent means of three replicates ± standard deviations. Similar results were obtained in at least three independent experiments. (B) Promoter activity of the rsmZ gene in WT, gacS , and gacA was determined by β-galactosidase activities. Treatments included strains with plasmid pKT-2 containing either a promoter-less lacZ insertion or the rsmZ promoter fused to the promoter-less lacZ gene (P rsmZ -lacZ ). Insertions were made in front of the promoter-less gfp gene, already present on the plasmid. Bacterial strains were grown in LB medium for 24 h at 28°C with shaking to an OD 600 of ∼1.8. Each value is the average from two different cultures ± standard deviations with two independent experiments. The experiment was repeated at least twice and similar results were obtained. (C) Effect of rsmZ on AHL production. Total AHL extractions were prepared from cell-free supernatants of WT, Δ gacS , or Δ gacA with plasmid pLAFR3 containing either NI or P tac - rsmZ . Cell-free supernatants were made from overnight cultures grown in LB medium (OD 600 ∼1.8). The relative amount of AHL in each extract was determined by β-galactosidase assays using the AHL-specific reporter 30-84I/Z ( phzI − , phzB::lacZ ). Bars represent β-galactosidase activity measured in Miller Units of 30-84I/Z grown with the AHL extracts indicated. Each value is the average from three different cultures ± standard deviations. (D) Relative expression of phzI , phzR , and phzY genes in the gacA mutant (harboring the pLAFR3-P tac - rsmZ ) compared with WT (harboring pLAFR3 empty vector) as determined by qPCR. rpoD was used as the reference gene. Cells were grown in AB minimal medium + 2% casamino acid for 18 h with shaking to an OD 600 of 1.2. Data points represent means of three replicates ± standard deviations. These experiments were repeated at least three times and similar results were obtained.

    Techniques Used: In Vitro, Plasmid Preparation, Activity Assay, Expressing, Mutagenesis, Real-time Polymerase Chain Reaction

    Verification of RNA-seq data by quantitative reverse transcription-polymerase chase reaction (qPCR). The relative fold change of gacA , gacS , rpoS , iopB , rpeB , pip , phzI , phzR , and phzB genes in gacA mutant compared towith the WT strain. The gene rpoD was used as the reference. Cells were grown in AB minimal medium + 2% casamino acid for 18 h with shaking to an OD 600 of 1.2. Data points represent means of three replicates ± standard deviations. These experiments were repeated at least three times and similar results were obtained.
    Figure Legend Snippet: Verification of RNA-seq data by quantitative reverse transcription-polymerase chase reaction (qPCR). The relative fold change of gacA , gacS , rpoS , iopB , rpeB , pip , phzI , phzR , and phzB genes in gacA mutant compared towith the WT strain. The gene rpoD was used as the reference. Cells were grown in AB minimal medium + 2% casamino acid for 18 h with shaking to an OD 600 of 1.2. Data points represent means of three replicates ± standard deviations. These experiments were repeated at least three times and similar results were obtained.

    Techniques Used: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Mutagenesis

    9) Product Images from "Antiviral therapy may decrease HBx, affecting cccDNA and MSL2 in hepatocarcinogenesis"

    Article Title: Antiviral therapy may decrease HBx, affecting cccDNA and MSL2 in hepatocarcinogenesis

    Journal: Oncology Letters

    doi: 10.3892/ol.2019.10833

    cccDNA detection using quantitative PCR (qPCR)
    Figure Legend Snippet: cccDNA detection using quantitative PCR (qPCR)

    Techniques Used: Real-time Polymerase Chain Reaction

    10) Product Images from "A Reverse Time-Course Method for Transcriptional Chase Analyses of mRNA Half-Lives in Cultured Cells"

    Article Title: A Reverse Time-Course Method for Transcriptional Chase Analyses of mRNA Half-Lives in Cultured Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0040827

    Comparative transcriptional chase analyses of a long-lived test mRNA. (A) Aggregate analysis using the conventional method. Cell aliquots were amended with dox at t 0 , and sacrificed at defined intervals. Levels of β WT mRNA were determined by RT-qPCR relative to control dox-indifferent β-actin mRNA, using the ΔΔCt method. Normalized RT-qPCR values for β WT mRNA were corrected for aliquot-specific cell numbers, then plotted. Points represent the mean ± S.D. from three replicate experiments. A t 1/2 value was calculated from the exponential decay constant corresponding to the best-fit curve. (B) Aggregate analysis using the reverse-chase method. Cell aliquots were amended with dox at defined intervals and sacrificed simultaneously at t = 80 h. Normalized values for β WT mRNA were determined by RT-qPCR, then plotted. Points represent the mean ± S.D. from three replicate experiments. A t 1/2 value was calculated from the exponential decay constant corresponding to the best-fit curve, corrected for an expansion factor describing the growth rate of cultured HeLa cells. (C) Analyses of individual replicates using the conventional method. Normalized values for each of three biological replicates reported in panel A were corrected for the number of cells present in each aliquot at the time of sacrifice, and t 1/2 values calculated. (D) Analyses of individual replicates using the reverse-chase method. Normalized values for each of three biological replicates reported in panel B were directly plotted, and t 1/2 values calculated following correction for interval cell expansion.
    Figure Legend Snippet: Comparative transcriptional chase analyses of a long-lived test mRNA. (A) Aggregate analysis using the conventional method. Cell aliquots were amended with dox at t 0 , and sacrificed at defined intervals. Levels of β WT mRNA were determined by RT-qPCR relative to control dox-indifferent β-actin mRNA, using the ΔΔCt method. Normalized RT-qPCR values for β WT mRNA were corrected for aliquot-specific cell numbers, then plotted. Points represent the mean ± S.D. from three replicate experiments. A t 1/2 value was calculated from the exponential decay constant corresponding to the best-fit curve. (B) Aggregate analysis using the reverse-chase method. Cell aliquots were amended with dox at defined intervals and sacrificed simultaneously at t = 80 h. Normalized values for β WT mRNA were determined by RT-qPCR, then plotted. Points represent the mean ± S.D. from three replicate experiments. A t 1/2 value was calculated from the exponential decay constant corresponding to the best-fit curve, corrected for an expansion factor describing the growth rate of cultured HeLa cells. (C) Analyses of individual replicates using the conventional method. Normalized values for each of three biological replicates reported in panel A were corrected for the number of cells present in each aliquot at the time of sacrifice, and t 1/2 values calculated. (D) Analyses of individual replicates using the reverse-chase method. Normalized values for each of three biological replicates reported in panel B were directly plotted, and t 1/2 values calculated following correction for interval cell expansion.

    Techniques Used: Quantitative RT-PCR, Cell Culture

    11) Product Images from "miR‐515‐5p controls cancer cell migration through MARK4 regulation"

    Article Title: miR‐515‐5p controls cancer cell migration through MARK4 regulation

    Journal: EMBO Reports

    doi: 10.15252/embr.201540970

    miR‐515‐5p regulates mRNA s involved in cell migration RNA ‐seq of MCF 7 and MDA ‐ MB ‐231 transfected with miR‐515‐5p revealed the down‐regulation of five transcripts, NRAS , FZD 4, CDC 42 BPA , PIK 3C2B and MARK 4. The effect of miR‐515‐5p overexpression on NRAS , FZD 4, CDC 42 BPA , PIK 3C2B and MARK 4 mRNA levels in MCF 7 (B), MDA ‐ MB ‐231(C), A549 and H1299 (D) was determined by qPCR 48 h following transfection of the indicated cell line with non‐targeting control miR ( NC ) or miR‐515‐5p. Data are displayed as the normalised mean ± SEM of n = 4. P ‐values were calculated by t ‐test between mi RNA conditions and their respective NC conditions (* P
    Figure Legend Snippet: miR‐515‐5p regulates mRNA s involved in cell migration RNA ‐seq of MCF 7 and MDA ‐ MB ‐231 transfected with miR‐515‐5p revealed the down‐regulation of five transcripts, NRAS , FZD 4, CDC 42 BPA , PIK 3C2B and MARK 4. The effect of miR‐515‐5p overexpression on NRAS , FZD 4, CDC 42 BPA , PIK 3C2B and MARK 4 mRNA levels in MCF 7 (B), MDA ‐ MB ‐231(C), A549 and H1299 (D) was determined by qPCR 48 h following transfection of the indicated cell line with non‐targeting control miR ( NC ) or miR‐515‐5p. Data are displayed as the normalised mean ± SEM of n = 4. P ‐values were calculated by t ‐test between mi RNA conditions and their respective NC conditions (* P

    Techniques Used: Migration, RNA Sequencing Assay, Multiple Displacement Amplification, Transfection, Over Expression, Real-time Polymerase Chain Reaction

    12) Product Images from "The Characterization of RNA Viruses in Tropical Seawater Using Targeted PCR and Metagenomics"

    Article Title: The Characterization of RNA Viruses in Tropical Seawater Using Targeted PCR and Metagenomics

    Journal: mBio

    doi: 10.1128/mBio.01210-14

    Distribution of picornavirad-like viruses in a CsCl buoyant density gradient. (Upper panel) The total RNA from the 2009 sample measured in each fraction of a buoyant density gradient (redrawn using data from reference 4 ) is shown along with the average amplification signal from endpoint PCR with degenerate primers targeting marine picornavirad-like viruses. The amplification signal value (in arbitrary relative fluorescence units) was determined by image analysis of the amplicons on an agarose gel and is presented as the running average of the signal for two sets of primers broadly targeting marine picornavirad-like subclade 1 (Mplsc1) and Mplsc2. (Lower panel) The copy numbers of two specific RdRp phylotypes that were identified by cloning and sequencing of amplicons from two of the fractions as determined by RT-qPCR. The shaded regions in both panels indicate the fractions used to create clone libraries and are labeled according to the phylotype (A or B) that was found only in that library.
    Figure Legend Snippet: Distribution of picornavirad-like viruses in a CsCl buoyant density gradient. (Upper panel) The total RNA from the 2009 sample measured in each fraction of a buoyant density gradient (redrawn using data from reference 4 ) is shown along with the average amplification signal from endpoint PCR with degenerate primers targeting marine picornavirad-like viruses. The amplification signal value (in arbitrary relative fluorescence units) was determined by image analysis of the amplicons on an agarose gel and is presented as the running average of the signal for two sets of primers broadly targeting marine picornavirad-like subclade 1 (Mplsc1) and Mplsc2. (Lower panel) The copy numbers of two specific RdRp phylotypes that were identified by cloning and sequencing of amplicons from two of the fractions as determined by RT-qPCR. The shaded regions in both panels indicate the fractions used to create clone libraries and are labeled according to the phylotype (A or B) that was found only in that library.

    Techniques Used: Amplification, Polymerase Chain Reaction, Fluorescence, Agarose Gel Electrophoresis, Clone Assay, Sequencing, Quantitative RT-PCR, Labeling

    13) Product Images from "MOF-associated complexes ensure stem cell identity and Xist repression"

    Article Title: MOF-associated complexes ensure stem cell identity and Xist repression

    Journal: eLife

    doi: 10.7554/eLife.02024

    Assessment of ChIP signals around the TSSs of putative target genes as determined by ChIP-seq. ( A ) Genome Browser snapshots of several MSL/NSL (first three from the left) or NSL-only target genes and respective sequencing-depth-normalized ChIP-seq and input signals from ESCs. The exact genomic coordinates are indicated on top of each panel. Gene names are indicated on the bottom. ( B ) ChIP-qPCR validation for MOF (green), KANSL3 (purple), MSL1 (blue) and MSL2 (red) signals. Immunoprecipitated DNA was amplified by qPCR with primer sets positioned at the promoter (P) and end (E) of the coding sequence ( Supplementary file 3A ). Results are expressed as mean ± SD of three biological replicates; cells were harvested for experiments on day 4 ( Kansl3, Msl1, Msl2 ) or 5 ( Mof ) of knockdown. ( C ) ChIP-qPCR for MSL1 (blue), MSL2 (red) and KANSL3 (purple) in ESCs treated with sh-RNA (scrambled or sh Mof ). Signals on genes were evaluated using primers at the promoter (P), and end (E) of the coding sequence. Results are expressed as mean ± SD of three biological replicates; cells were harvested for experiments on day 5 of Mof knockdown. DOI: http://dx.doi.org/10.7554/eLife.02024.012
    Figure Legend Snippet: Assessment of ChIP signals around the TSSs of putative target genes as determined by ChIP-seq. ( A ) Genome Browser snapshots of several MSL/NSL (first three from the left) or NSL-only target genes and respective sequencing-depth-normalized ChIP-seq and input signals from ESCs. The exact genomic coordinates are indicated on top of each panel. Gene names are indicated on the bottom. ( B ) ChIP-qPCR validation for MOF (green), KANSL3 (purple), MSL1 (blue) and MSL2 (red) signals. Immunoprecipitated DNA was amplified by qPCR with primer sets positioned at the promoter (P) and end (E) of the coding sequence ( Supplementary file 3A ). Results are expressed as mean ± SD of three biological replicates; cells were harvested for experiments on day 4 ( Kansl3, Msl1, Msl2 ) or 5 ( Mof ) of knockdown. ( C ) ChIP-qPCR for MSL1 (blue), MSL2 (red) and KANSL3 (purple) in ESCs treated with sh-RNA (scrambled or sh Mof ). Signals on genes were evaluated using primers at the promoter (P), and end (E) of the coding sequence. Results are expressed as mean ± SD of three biological replicates; cells were harvested for experiments on day 5 of Mof knockdown. DOI: http://dx.doi.org/10.7554/eLife.02024.012

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, Real-time Polymerase Chain Reaction, Immunoprecipitation, Amplification

    14) Product Images from "Systematic Analysis of tRNA-Derived Small RNAs Reveals Novel Potential Therapeutic Targets of Traditional Chinese Medicine (Buyang-Huanwu-Decoction) on Intracerebral Hemorrhage"

    Article Title: Systematic Analysis of tRNA-Derived Small RNAs Reveals Novel Potential Therapeutic Targets of Traditional Chinese Medicine (Buyang-Huanwu-Decoction) on Intracerebral Hemorrhage

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.29744

    Study design illustration. ICH, intracerebral hemorrhage; BYHWD, Buyang-Huanwu-Decoction; FC, fold change; qPCR, quantitative real-time PCR.
    Figure Legend Snippet: Study design illustration. ICH, intracerebral hemorrhage; BYHWD, Buyang-Huanwu-Decoction; FC, fold change; qPCR, quantitative real-time PCR.

    Techniques Used: Real-time Polymerase Chain Reaction

    15) Product Images from "Downregulation of TACC3 inhibits tumor growth and migration in osteosarcoma cells through regulation of the NF-κB signaling pathway"

    Article Title: Downregulation of TACC3 inhibits tumor growth and migration in osteosarcoma cells through regulation of the NF-κB signaling pathway

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.8262

    (A) mRNA level of TACC3 was examined in osteosarcoma cell lines using RT-qPCR. (B) Protein levels of TACC3 were determined by western blot analysis in various osteosarcoma cell lines. (C) The mRNA level of TACC3 in osteosarcoma tissues and normal matched non-cancerous tissue as assessed by RT-qPCR. NS, non-cancerous tissue; OS, osteosarcoma; TACC3, transforming acidic coiled-coil protein; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
    Figure Legend Snippet: (A) mRNA level of TACC3 was examined in osteosarcoma cell lines using RT-qPCR. (B) Protein levels of TACC3 were determined by western blot analysis in various osteosarcoma cell lines. (C) The mRNA level of TACC3 in osteosarcoma tissues and normal matched non-cancerous tissue as assessed by RT-qPCR. NS, non-cancerous tissue; OS, osteosarcoma; TACC3, transforming acidic coiled-coil protein; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

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

    16) Product Images from "Rab40b regulates trafficking of MMP2 and MMP9 during invadopodia formation and invasion of breast cancer cells"

    Article Title: Rab40b regulates trafficking of MMP2 and MMP9 during invadopodia formation and invasion of breast cancer cells

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.126573

    Rab40b is required for invadopodia-associated ECM degradation. (A–F) Untreated or Rab40b-siRNA-treated MDA-MB-231 cells were plated on gelatin and fibronectin HiLyte Fluor488-coated coverslips (D–F). After 20 hours of incubation, cells were fixed and stained with Rhodamine-phalloidin (A–C). Scale bars: 25 µm. (G) Quantification of ECM degradation in untreated and Rab40b-siRNA-treated MDA-MB-231 cells. Data shown are the means and s.e. of three independent experiments. n is the total number of cells analyzed. (H) Quantification of ECM degradation in MDA-MB-231 cells treated with either GM6001 (broad-spectrum MMP inhibitor) or SB3CT (MMP2/MMP9 inhibitor). Where indicated, cells were also treated with Rab40b siRNA. Data shown are the means and s.e. of three independent experiments. n is the total number of cells analyzed. (I) Mock-, Rab40b-siRNA- or VAMP4 siRNA-treated cells were harvested and the levels of mRNA encoding Rab40b analyzed by qPCR (graph). The data shown are the means and s.d. The levels of VAMP4 were analyzed by western blotting (top panels) with tubulin used as a loading control. (J) Quantification of ECM degradation in mock-, Rab40b-siRNA- or VAMP4-siRNA-treated MDA-MB-231 cells. Data shown are the means and s.e. of three independent experiments. n is the total number of cells analyzed.
    Figure Legend Snippet: Rab40b is required for invadopodia-associated ECM degradation. (A–F) Untreated or Rab40b-siRNA-treated MDA-MB-231 cells were plated on gelatin and fibronectin HiLyte Fluor488-coated coverslips (D–F). After 20 hours of incubation, cells were fixed and stained with Rhodamine-phalloidin (A–C). Scale bars: 25 µm. (G) Quantification of ECM degradation in untreated and Rab40b-siRNA-treated MDA-MB-231 cells. Data shown are the means and s.e. of three independent experiments. n is the total number of cells analyzed. (H) Quantification of ECM degradation in MDA-MB-231 cells treated with either GM6001 (broad-spectrum MMP inhibitor) or SB3CT (MMP2/MMP9 inhibitor). Where indicated, cells were also treated with Rab40b siRNA. Data shown are the means and s.e. of three independent experiments. n is the total number of cells analyzed. (I) Mock-, Rab40b-siRNA- or VAMP4 siRNA-treated cells were harvested and the levels of mRNA encoding Rab40b analyzed by qPCR (graph). The data shown are the means and s.d. The levels of VAMP4 were analyzed by western blotting (top panels) with tubulin used as a loading control. (J) Quantification of ECM degradation in mock-, Rab40b-siRNA- or VAMP4-siRNA-treated MDA-MB-231 cells. Data shown are the means and s.e. of three independent experiments. n is the total number of cells analyzed.

    Techniques Used: Multiple Displacement Amplification, Incubation, Staining, Real-time Polymerase Chain Reaction, Western Blot

    Rab40b knockdown decreases MMP2 and MMP9 secretion in MDA-MB-231 cells. (A) Schematic representation of Rab40b domain structure. (B) The efficiency of Rab40b knockdown as determined by qPCR. (C,D) MDA-MB-231 cells expressing MMP2–Myc (C) or MMP9–Myc (D) were transfected with four different Rab40b siRNAs. Two days later, equal number of cells were plated in six-well dishes and incubated with 1 ml of medium for 24 hours. Medium was then collected and the effect of Rab40b knockdown on secretion of MMP2–Myc and MMP9–Myc analyzed by western blotting. Data shown are the means and s.d. of three independent experiments. (E) MDA-MB-231 cells were transfected with Rab40b siRNA#2. Two days later, equal number of cells were plated in six-well dishes and incubated with 1 ml of Opti-MEM for 24 hours. Opti-MEM was then collected and the effect of Rab40b knockdown on secretion of endogenous MMP2 and MMP9 was analyzed by zymography. Fetal bovine serum (rich in secreted MMP2/9) in the first lane was used as a positive control. Opti-MEM collected from a six-well dish without cells was used as negative control. (F) Mock-, Rab40b-siRNA- or VAMP4-siRNA-treated MDA-MB-231 cells were harvested and analyzed by flow cytometry to measure the levels of endogenous plasma membrane MT1-MMP. Data shown are the means and s.d. of three independent experiments. (G) Mock- or Rab40b-siRNA-treated MDA-MB-231 cells were plated in six-well dishes and stimulated with 1 mg/ml of LPS. After incubation for 16 hours, medium was collected and the levels of secreted IL-6 analyzed using ELISA. The data shown are the means and s.d. of three independent experiments.
    Figure Legend Snippet: Rab40b knockdown decreases MMP2 and MMP9 secretion in MDA-MB-231 cells. (A) Schematic representation of Rab40b domain structure. (B) The efficiency of Rab40b knockdown as determined by qPCR. (C,D) MDA-MB-231 cells expressing MMP2–Myc (C) or MMP9–Myc (D) were transfected with four different Rab40b siRNAs. Two days later, equal number of cells were plated in six-well dishes and incubated with 1 ml of medium for 24 hours. Medium was then collected and the effect of Rab40b knockdown on secretion of MMP2–Myc and MMP9–Myc analyzed by western blotting. Data shown are the means and s.d. of three independent experiments. (E) MDA-MB-231 cells were transfected with Rab40b siRNA#2. Two days later, equal number of cells were plated in six-well dishes and incubated with 1 ml of Opti-MEM for 24 hours. Opti-MEM was then collected and the effect of Rab40b knockdown on secretion of endogenous MMP2 and MMP9 was analyzed by zymography. Fetal bovine serum (rich in secreted MMP2/9) in the first lane was used as a positive control. Opti-MEM collected from a six-well dish without cells was used as negative control. (F) Mock-, Rab40b-siRNA- or VAMP4-siRNA-treated MDA-MB-231 cells were harvested and analyzed by flow cytometry to measure the levels of endogenous plasma membrane MT1-MMP. Data shown are the means and s.d. of three independent experiments. (G) Mock- or Rab40b-siRNA-treated MDA-MB-231 cells were plated in six-well dishes and stimulated with 1 mg/ml of LPS. After incubation for 16 hours, medium was collected and the levels of secreted IL-6 analyzed using ELISA. The data shown are the means and s.d. of three independent experiments.

    Techniques Used: Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Expressing, Transfection, Incubation, Western Blot, Zymography, Positive Control, Negative Control, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    17) Product Images from "In vitro activity of aryl-thiazole derivatives against Schistosoma mansoni schistosomula and adult worms"

    Article Title: In vitro activity of aryl-thiazole derivatives against Schistosoma mansoni schistosomula and adult worms

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0225425

    Vitellaria and female reproduction gene expression inhibition in adult females upon NJ05 treatment. Adult couples were treated with NJ05 (25 μM) or with vehicle DMSO (0.1%) for 2 days. The parasites were stored in RNAlater (Ambion) for further processing. All parasite couples were separated and only the females had their RNA extracted followed by cDNA synthesis. The genes measured by RT-qPCR were Smp_131110 (p14), Smp_000430 (Egg Shell Protein (ESP)), Smp_013540 (Tyrosinase 2 (Tyr 2)), Smp_014610 (p48), Smp_000290 (fs800), Smp_055740 (Nanos 2). The geometric mean from two reference genes (Smp_090920 and Smp_123610) was used for normalization with the DCT method. The plotted data is retrieved from DDCT analyses in which the DMSO-treated sample is the control. Significant fold-change in gene expression between DMSO and NJ05 treatment is shown by the numbers inside the brackets. Mean ± SEM of four replicates is shown. Student unpaired parametric two-sided t-test was used, and statistically significant differences are represented by the asterisks. *p ≤ 0.05; **p ≤ 0.01.
    Figure Legend Snippet: Vitellaria and female reproduction gene expression inhibition in adult females upon NJ05 treatment. Adult couples were treated with NJ05 (25 μM) or with vehicle DMSO (0.1%) for 2 days. The parasites were stored in RNAlater (Ambion) for further processing. All parasite couples were separated and only the females had their RNA extracted followed by cDNA synthesis. The genes measured by RT-qPCR were Smp_131110 (p14), Smp_000430 (Egg Shell Protein (ESP)), Smp_013540 (Tyrosinase 2 (Tyr 2)), Smp_014610 (p48), Smp_000290 (fs800), Smp_055740 (Nanos 2). The geometric mean from two reference genes (Smp_090920 and Smp_123610) was used for normalization with the DCT method. The plotted data is retrieved from DDCT analyses in which the DMSO-treated sample is the control. Significant fold-change in gene expression between DMSO and NJ05 treatment is shown by the numbers inside the brackets. Mean ± SEM of four replicates is shown. Student unpaired parametric two-sided t-test was used, and statistically significant differences are represented by the asterisks. *p ≤ 0.05; **p ≤ 0.01.

    Techniques Used: Expressing, Inhibition, Quantitative RT-PCR, End-sequence Profiling

    18) Product Images from "Genotypic Identification of Cystoisospora in Immunocompromised Patients Using Tm-Variation Analysis"

    Article Title: Genotypic Identification of Cystoisospora in Immunocompromised Patients Using Tm-Variation Analysis

    Journal: The Korean Journal of Parasitology

    doi: 10.3347/kjp.2017.55.6.601

    Agarose gel electrophoresis of RFLP profiles of ITS2 qPCR amplified Cystoisospora products using ALU I restriction enzyme. Lane M, 50 bp molecular weight marker; lane 1, confirmed sample (genotype II: 130 bp); lane 2, confirmed sample (genotype I: 67bp); lane 3, control.
    Figure Legend Snippet: Agarose gel electrophoresis of RFLP profiles of ITS2 qPCR amplified Cystoisospora products using ALU I restriction enzyme. Lane M, 50 bp molecular weight marker; lane 1, confirmed sample (genotype II: 130 bp); lane 2, confirmed sample (genotype I: 67bp); lane 3, control.

    Techniques Used: Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    19) Product Images from "Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells"

    Article Title: Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells

    Journal: Oncology Reports

    doi: 10.3892/or.2018.6329

    Induction of apoptosis in A375 cells by Licochalcone D (LD) treatment. (A) Cell morphological changes were observed by phase-contrast microscopy (magnification, ×200) after treatment with LD (0, 30, 60 and 90 µmol/l) for 24 h. (B) Apoptosis was visualized by the appropriate changes in nuclei stained with Hoechst 33258 (blue) (magnification, ×200). (C) The effects of LD on the induction of A375 cell apoptosis were analyzed by FCM analysis. (D) The apoptosis rate as statistically analyzed. (E) RT-PCR analyses of A375 cells to evaluate mRNA expression of Bcl-2, Bax, caspase-3 and caspase-9. (F) qPCR analyses of A375 cells to evaluate mRNA expression of Bcl-2, Bax, caspase-3 and caspase-9, and relative intensities were normalized by levels of GAPDH. The untreated group level was considered as ‘1.0’. Data are presented as means ± SD of at least three independent experiments. *P
    Figure Legend Snippet: Induction of apoptosis in A375 cells by Licochalcone D (LD) treatment. (A) Cell morphological changes were observed by phase-contrast microscopy (magnification, ×200) after treatment with LD (0, 30, 60 and 90 µmol/l) for 24 h. (B) Apoptosis was visualized by the appropriate changes in nuclei stained with Hoechst 33258 (blue) (magnification, ×200). (C) The effects of LD on the induction of A375 cell apoptosis were analyzed by FCM analysis. (D) The apoptosis rate as statistically analyzed. (E) RT-PCR analyses of A375 cells to evaluate mRNA expression of Bcl-2, Bax, caspase-3 and caspase-9. (F) qPCR analyses of A375 cells to evaluate mRNA expression of Bcl-2, Bax, caspase-3 and caspase-9, and relative intensities were normalized by levels of GAPDH. The untreated group level was considered as ‘1.0’. Data are presented as means ± SD of at least three independent experiments. *P

    Techniques Used: Microscopy, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

    20) Product Images from "Dengue Virus Infects Primary Human Hair Follicle Dermal Papilla Cells"

    Article Title: Dengue Virus Infects Primary Human Hair Follicle Dermal Papilla Cells

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00268

    DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 (STAT1), and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P
    Figure Legend Snippet: DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 (STAT1), and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P

    Techniques Used: Expressing, Quantitative RT-PCR, Infection

    21) Product Images from "Insect Neuropeptide Bursicon Homodimers Induce Innate Immune and Stress Genes during Molting by Activating the NF-?B Transcription Factor Relish"

    Article Title: Insect Neuropeptide Bursicon Homodimers Induce Innate Immune and Stress Genes during Molting by Activating the NF-?B Transcription Factor Relish

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034510

    Burs α−α and burs β−β treatments induced expression of AMP genes and suppression of bacterial growth in adults. Separate groups of adult flies were neck-ligated immediately after emergence and at 1 h post-ligation injected with 60 ng/0.5 µl of r-burs α−β, burs α−α, or burs β−β. The control flies were injected with the purified cell culture transfected with blank pcDNA 3.1 vector. After the indicated incubation periods, expression of the AMP genes was determined by qPCR. (A) AMP transcript levels in wild type adults. For bacterial inhibition assay (B), neck-ligated wild type flies were injected with r-burs α−β heterodimer, burs α−α homodimer, burs β−β homodimer or blank vector transfected sample, respectively, homogenized and centrifuged for 15 min at 16,000 g. The resulting supernatants were challenged with indicated titers of E. coli for 6 h before plating for colony count. The histograms show the means ± SEM, n = 3 biologically independent experiments.
    Figure Legend Snippet: Burs α−α and burs β−β treatments induced expression of AMP genes and suppression of bacterial growth in adults. Separate groups of adult flies were neck-ligated immediately after emergence and at 1 h post-ligation injected with 60 ng/0.5 µl of r-burs α−β, burs α−α, or burs β−β. The control flies were injected with the purified cell culture transfected with blank pcDNA 3.1 vector. After the indicated incubation periods, expression of the AMP genes was determined by qPCR. (A) AMP transcript levels in wild type adults. For bacterial inhibition assay (B), neck-ligated wild type flies were injected with r-burs α−β heterodimer, burs α−α homodimer, burs β−β homodimer or blank vector transfected sample, respectively, homogenized and centrifuged for 15 min at 16,000 g. The resulting supernatants were challenged with indicated titers of E. coli for 6 h before plating for colony count. The histograms show the means ± SEM, n = 3 biologically independent experiments.

    Techniques Used: Expressing, Ligation, Injection, Purification, Cell Culture, Transfection, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Inhibition

    Transcription expression profiles of burs α and burs β subunits and AMP genes in pharate and newly-emerged adults. qPCR analysis (A) of bursicon α and β gene expression in pharate (PA) and eclosed adults (A) 0–24 h after eclosion; qPCR analysis (B) of AMP gene expression in pharate and eclosed adults 0–24 h after eclosion; Transcription expression ( C ) of AMP genes in 24 h-old adults injected with blank vector transfected sample, r-burs α−α homodimer, r-burs β−β homodimer and r-burs α−β heterodimer, respectively.
    Figure Legend Snippet: Transcription expression profiles of burs α and burs β subunits and AMP genes in pharate and newly-emerged adults. qPCR analysis (A) of bursicon α and β gene expression in pharate (PA) and eclosed adults (A) 0–24 h after eclosion; qPCR analysis (B) of AMP gene expression in pharate and eclosed adults 0–24 h after eclosion; Transcription expression ( C ) of AMP genes in 24 h-old adults injected with blank vector transfected sample, r-burs α−α homodimer, r-burs β−β homodimer and r-burs α−β heterodimer, respectively.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Injection, Plasmid Preparation, Transfection

    AMP transcript levels in rk 4 mutant. Newly emerged adult flies were neck-ligated immediately after emergence and at 1 h post-ligation injected with r-burs α−β heterodimer, burs α−α homodimer, burs β−β homodimer or blank vector transfected sample, as described in Figure 3 . RNA was extracted for qPCR analysis of 11 representative genes (A). Larva FB from the mutant was also used to assay the effect of mutation on burs α−α and burs β−β homodimer induced AMP expression (B). The histograms show the means ± SEM, n = 3 biologically independent experiments.
    Figure Legend Snippet: AMP transcript levels in rk 4 mutant. Newly emerged adult flies were neck-ligated immediately after emergence and at 1 h post-ligation injected with r-burs α−β heterodimer, burs α−α homodimer, burs β−β homodimer or blank vector transfected sample, as described in Figure 3 . RNA was extracted for qPCR analysis of 11 representative genes (A). Larva FB from the mutant was also used to assay the effect of mutation on burs α−α and burs β−β homodimer induced AMP expression (B). The histograms show the means ± SEM, n = 3 biologically independent experiments.

    Techniques Used: Mutagenesis, Ligation, Injection, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Expressing

    Burs α−α and burs β−β treatments induced expression of AMP genes and suppression of bacterial growth in larva FB. FB was dissected from early wondering 3 rd instar larvae and incubated with r-burs α−α or burs β−β homodimer or r-burs α+β heterodimer for 0.5, 1 and 3 h. The control group received the blank vector transfected sample. After incubation, RNA was extracted for qPCR analysis of 4 representative AMP genes (A). For bacterial inhibition assay, FB was homogenized and centrifuged at 16000 g for 20 min at 4°C. The resulting supernatants were challenged with indicated titers of E. coli for 6 h before plating for colony count (B). The histograms show the means ± SEM, n = 3 biologically independent experiments.
    Figure Legend Snippet: Burs α−α and burs β−β treatments induced expression of AMP genes and suppression of bacterial growth in larva FB. FB was dissected from early wondering 3 rd instar larvae and incubated with r-burs α−α or burs β−β homodimer or r-burs α+β heterodimer for 0.5, 1 and 3 h. The control group received the blank vector transfected sample. After incubation, RNA was extracted for qPCR analysis of 4 representative AMP genes (A). For bacterial inhibition assay, FB was homogenized and centrifuged at 16000 g for 20 min at 4°C. The resulting supernatants were challenged with indicated titers of E. coli for 6 h before plating for colony count (B). The histograms show the means ± SEM, n = 3 biologically independent experiments.

    Techniques Used: Expressing, Incubation, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Inhibition

    22) Product Images from "Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer"

    Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr3232

    The effect of PIP knockdown on cell invasion and viability . (A) qPCR to demonstrate PIP-knockdown efficiencies with siRNA-duplex1 (D1) and siRNA-duplex2 (D2) in MDA-MB-453 cell line. PIP expression following knockdown was assessed relative to non-targeting siRNA control (CTL) and fold change is shown for each duplex. (B) Western blot analysis to show PIP protein level following PIP-knockdown in MDA-MB-453 cell line as described in (A). Fold changes (RR) in band densities were measured relative to the control (CTL). (C) The effect of PIP expression on cell invasion. Cell invasion assays were carried out after PIP-knockdown with PIP-D1 and PIP-D2 in MDA-MB-453 cell line. Transfection with non-targeting siRNA control (CTL) was used as a control. *, P
    Figure Legend Snippet: The effect of PIP knockdown on cell invasion and viability . (A) qPCR to demonstrate PIP-knockdown efficiencies with siRNA-duplex1 (D1) and siRNA-duplex2 (D2) in MDA-MB-453 cell line. PIP expression following knockdown was assessed relative to non-targeting siRNA control (CTL) and fold change is shown for each duplex. (B) Western blot analysis to show PIP protein level following PIP-knockdown in MDA-MB-453 cell line as described in (A). Fold changes (RR) in band densities were measured relative to the control (CTL). (C) The effect of PIP expression on cell invasion. Cell invasion assays were carried out after PIP-knockdown with PIP-D1 and PIP-D2 in MDA-MB-453 cell line. Transfection with non-targeting siRNA control (CTL) was used as a control. *, P

    Techniques Used: Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Expressing, CTL Assay, Western Blot, Transfection

    The regulation of molecular apocrine genes by the AR-ERK feedback loop . (A) Heat map of top ranking molecular apocrine-signature genes following the inhibition of AR-ERK signaling using qPCR data. Heat map shows fold changes for gene expression relative to control in MDA-MB-453 and HCC-1954 cell lines. Treatments were carried out by CI-1040 (CI) at 2 µM and 10 µM concentrations, flutamide (FLU) at 25 nM and 40 nM concentrations, and the combination of flutamide at 25 nM or 40 nM and CI-1040 at 2 µM concentrations. Red and green colors depict up-regulation and down-regulation, respectively. Bar indicates the range of fold changes in gene expression. (B) Box plots to demonstrate relative expression of PIP to control following AR-ERK inhibition in MDA-MB-453 and HCC-1954 cell lines using qPCR. CTL: control. (C) Western blot analysis to assess PIP protein levels following AR-ERK inhibition in MDA-MB-453 and HCC-1954 cell lines. Fold changes (RR) in band densities were measured relative to the control (CTL). AR, androgen receptor; ERK, extracellular signal-regulated kinase; qPCR, quantitative PCR; RR, relative risk.
    Figure Legend Snippet: The regulation of molecular apocrine genes by the AR-ERK feedback loop . (A) Heat map of top ranking molecular apocrine-signature genes following the inhibition of AR-ERK signaling using qPCR data. Heat map shows fold changes for gene expression relative to control in MDA-MB-453 and HCC-1954 cell lines. Treatments were carried out by CI-1040 (CI) at 2 µM and 10 µM concentrations, flutamide (FLU) at 25 nM and 40 nM concentrations, and the combination of flutamide at 25 nM or 40 nM and CI-1040 at 2 µM concentrations. Red and green colors depict up-regulation and down-regulation, respectively. Bar indicates the range of fold changes in gene expression. (B) Box plots to demonstrate relative expression of PIP to control following AR-ERK inhibition in MDA-MB-453 and HCC-1954 cell lines using qPCR. CTL: control. (C) Western blot analysis to assess PIP protein levels following AR-ERK inhibition in MDA-MB-453 and HCC-1954 cell lines. Fold changes (RR) in band densities were measured relative to the control (CTL). AR, androgen receptor; ERK, extracellular signal-regulated kinase; qPCR, quantitative PCR; RR, relative risk.

    Techniques Used: Inhibition, Real-time Polymerase Chain Reaction, Expressing, Multiple Displacement Amplification, CTL Assay, Western Blot

    23) Product Images from "Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix"

    Article Title: Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-16960-8

    Comparison of different storage temperature and trehalose for FTA Elute card stored for 2 days. ( A ) Untreated or 50 mg/ml trehalose-treated 6 mm FTA Elute card disc punch-out spotted with 20 µl serum were dried and stored at RT, 4 °C or −20 °C for 2 days before RNA was extracted using QIAzol lysis reagent. 10 miRNAs were quantified by RT-qPCR and their copy numbers were presented. Neat refers to copy number of each individual miRNA obtained from 20 µl serum extracted directly using QIAzol lysis reagent. The individual % miRNA recovery was compared between untreated and trehalose-treated FTA Elute card disc punch-out. ( B ) Average % miRNA recovery of all 10 miRNAs was calculated and plotted to compare the effect of pre-treatment of FTA Elute card disc punch-out with 50mg/ml trehalose. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data between selected pairs. Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; **P ≤ 0.01; * P ≤ 0.05, n = 3).
    Figure Legend Snippet: Comparison of different storage temperature and trehalose for FTA Elute card stored for 2 days. ( A ) Untreated or 50 mg/ml trehalose-treated 6 mm FTA Elute card disc punch-out spotted with 20 µl serum were dried and stored at RT, 4 °C or −20 °C for 2 days before RNA was extracted using QIAzol lysis reagent. 10 miRNAs were quantified by RT-qPCR and their copy numbers were presented. Neat refers to copy number of each individual miRNA obtained from 20 µl serum extracted directly using QIAzol lysis reagent. The individual % miRNA recovery was compared between untreated and trehalose-treated FTA Elute card disc punch-out. ( B ) Average % miRNA recovery of all 10 miRNAs was calculated and plotted to compare the effect of pre-treatment of FTA Elute card disc punch-out with 50mg/ml trehalose. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data between selected pairs. Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; **P ≤ 0.01; * P ≤ 0.05, n = 3).

    Techniques Used: Lysis, Quantitative RT-PCR

    miRNA recovery from serum-spotted FTA Elute cards using water and heat was inefficient. ( A ) RNA was extracted directly from 20 µl serum (black) or 20 µl plasma collected in EDTA-containing tube (plasma-EDTA) (white) using QIAzol lysis reagent. 10 miRNAs were quantified by RT-qPCR based on their copy numbers. RNA was extracted from a 6 mm FTA Elute card disc punch-out spotted with 20 µl serum (black) or 20 µl plasma-EDTA (white), using water and heating at 95 °C for 30 min. 10 miRNAs were quantified by RT-qPCR and their copy numbers ( B ) and % miRNA recovery ( C ) were presented. (% recovery = (Copy number of miRNA extracted from FTA Elute card disc punch-out/Copy number of miRNA extracted directly from serum) × 100%). Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns: not significant, Student’s t -test, n = 3).
    Figure Legend Snippet: miRNA recovery from serum-spotted FTA Elute cards using water and heat was inefficient. ( A ) RNA was extracted directly from 20 µl serum (black) or 20 µl plasma collected in EDTA-containing tube (plasma-EDTA) (white) using QIAzol lysis reagent. 10 miRNAs were quantified by RT-qPCR based on their copy numbers. RNA was extracted from a 6 mm FTA Elute card disc punch-out spotted with 20 µl serum (black) or 20 µl plasma-EDTA (white), using water and heating at 95 °C for 30 min. 10 miRNAs were quantified by RT-qPCR and their copy numbers ( B ) and % miRNA recovery ( C ) were presented. (% recovery = (Copy number of miRNA extracted from FTA Elute card disc punch-out/Copy number of miRNA extracted directly from serum) × 100%). Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns: not significant, Student’s t -test, n = 3).

    Techniques Used: Lysis, Quantitative RT-PCR

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    Article Snippet: .. RC21 and RC1 were replaced by Tag-ZK21 primer (5′-ggccgtcatggtggcgaataaCCTGACAACACTAAaATTGGTGC-3′) and Tag-ZK1 primer (5′-ggccgtcatggtggcgaataaAGGATCATAGGTGATGAAGAAAAGT-3′). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers’ instructions. qPCR amplification was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), in a PikoReal 96 Real-Time PCR system (ThermoFisher Scientific). .. The cycle conditions were uracil-DNA glycosylase (UDG) activation at 50 °C for 2 min, dual-lock DNA polymerase at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing at 55 °C for 15 sec, and extension at 72 °C for 1 min. An MR766 infectious clone [ ] was used to generate a standard curve, and was subject to the same strand specific RT-qPCR protocol.

    Article Title: Antiviral therapy may decrease HBx, affecting cccDNA and MSL2 in hepatocarcinogenesis
    Article Snippet: .. A total of 1 ml of cccDNA was then used for qPCR amplification (7500 Real-time PCR Instrument system; Applied Biosystems; Thermo Fisher Scientific, Inc.), which was performed using TOPreal™ qPCR PreMIX SYBR Green (Enzynomics). β-actin amplicons were used as the internal reference for subsequent PCR analysis. .. β-actin amplicons were used as the internal reference for subsequent PCR analysis.

    Article Title: Insect Neuropeptide Bursicon Homodimers Induce Innate Immune and Stress Genes during Molting by Activating the NF-?B Transcription Factor Relish
    Article Snippet: Gene specific primers ( ) were used for qPCR amplification of AMP genes. qPCR amplification and analysis were carried out on an Applied Biosystems (ABI) 7500 Fast Real-Time PCR System. .. The final reaction volume was 25 µl using ABI SYBR green Supermix (ABI).

    Article Title: S1P3 confers differential S1P migration by autoreactive and non-autoreactive immature B cells and is required for normal B cell development
    Article Snippet: .. Quantitative PCR amplification was performed using Platinum SYBR Green qPCR SuperMix-UDG(Invitrogen Life Technologies) and detected on an MJ ResearchDNA Engine Opticon 2 real-time PCR machine. ..

    Article Title: miR‐515‐5p controls cancer cell migration through MARK4 regulation
    Article Snippet: .. To analyse mRNA expression, both cDNA conversion and qPCR amplification were performed as recommended by Applied Biosystems using the High‐Capacity cDNA Reverse Transcription Kit and Fast SYBR® Green Master Mix, respectively. ..

    Article Title: PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein
    Article Snippet: .. A 200-ng sample of total RNA was reverse-transcribed with the Superscript III Reverse Transcriptase (Invitrogen), using 10 μ random hexamer primers (Roche) for the Superscript Reverse Transcriptase (Invitrogen), according to the manufacturer's instructions. qPCR amplification was performed with a 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA) using the Platinum SYBR Green qPCR Supermix-UDG with ROX (Invitrogen), using specific primers (200 n) as listed in . .. In each experiment, the housekeeping genes, PPIB and YWHAZ , were amplified as a reference standard for normalization.

    Article Title: Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells
    Article Snippet: .. RT-PCR products were resolved on a 1.5% agarose gel, stained with ethidium bromide, and the intensity was quantified by Gel-Pro analysis software (ImageJ software, version 1.49n; National Institutes of Health, Bethesda, MD, USA). qPCR amplification was conducted using SYBR-Green q-RT-PCR kit reagents (Fermentas) according to the manufacturer's instructions. ..

    Article Title: Roles of the Gac-Rsm pathway in the regulation of phenazine biosynthesis in Pseudomonas chlororaphis 30-84
    Article Snippet: .. Elimination of contaminating DNA was confirmed via qPCR amplification of the rpoD gene with SYBR green® dye on an ABI 9400HT PCR machine (Life Technologies, Carlsbad, CA). ..

    Microarray:

    Article Title: The miR-99 family regulates the DNA damage response through its target SNF2H
    Article Snippet: For microarray analysis, RNA was further purified using RNAeasy RNA cleanup kit (Qiagen). .. For qPCR validation, Poly A tailing and cDNA preparation of mature microRNAs was performed using the NCODE miRNA amplification system (Invitrogen). qPCR amplification was performed using forward primers identical to the mature miRNA sequence, and NCODE universal reverse primers with Sybr Green ER (Invitrogen).

    Incubation:

    Article Title: The Characterization of RNA Viruses in Tropical Seawater Using Targeted PCR and Metagenomics
    Article Snippet: The reaction mixtures were brought to 55°C for 60 min and to 70°C for 15 min as a final termination step and were then supplemented with 1 µl RNase H (Invitrogen Corporation) and incubated for 20 min at 37°C. .. The qPCR amplification was performed on a 7300 real-time PCR system (Applied Biosystems) with Power SYBR green PCR Mastermix (Applied Biosystems).

    Article Title: MOF-associated complexes ensure stem cell identity and Xist repression
    Article Snippet: After incubation, 50 μl of 50% slurry bead solution was added for another incubation period (2 hr), then beads were washed: four times for 15 min with RIPA lysis buffer, two times for 1 min with LiCl IP wash buffer (250 mM LiCl, 10 mM Tris–HCl pH 8.0, 1 mM EDTA, 0.5% NP-40, 0.5% DOC, filtered through 0.2 micron filter unit), two times for 1 min with TE buffer (1 mM Tris–HCl pH 8.0, 1 mM EDTA, filtered through 0.2 micron filter unit). .. Purified ChIPed DNA was subjected to qPCR amplification (Applied Biosystems, Carlsbad, CA).

    Expressing:

    Article Title: The miR-99 family regulates the DNA damage response through its target SNF2H
    Article Snippet: Paragraph title: miRNA expression analysis ... For qPCR validation, Poly A tailing and cDNA preparation of mature microRNAs was performed using the NCODE miRNA amplification system (Invitrogen). qPCR amplification was performed using forward primers identical to the mature miRNA sequence, and NCODE universal reverse primers with Sybr Green ER (Invitrogen).

    Article Title: miR‐515‐5p controls cancer cell migration through MARK4 regulation
    Article Snippet: .. To analyse mRNA expression, both cDNA conversion and qPCR amplification were performed as recommended by Applied Biosystems using the High‐Capacity cDNA Reverse Transcription Kit and Fast SYBR® Green Master Mix, respectively. ..

    Article Title: PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein
    Article Snippet: A 200-ng sample of total RNA was reverse-transcribed with the Superscript III Reverse Transcriptase (Invitrogen), using 10 μ random hexamer primers (Roche) for the Superscript Reverse Transcriptase (Invitrogen), according to the manufacturer's instructions. qPCR amplification was performed with a 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA) using the Platinum SYBR Green qPCR Supermix-UDG with ROX (Invitrogen), using specific primers (200 n) as listed in . .. The expression of each target gene was normalized to the expression of the housekeeping genes and presented relative to the corresponding siControl sample.

    Article Title: Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells
    Article Snippet: Semi-quantitative reverse transcription-PCR (RT-PCR) ( ) and quantitative real-time PCR (qPCR) ( ) were performed to examine the mRNA expression of Bax, Bcl-2, caspase-3, caspase-9, MMP-2 and MMP-9. .. RT-PCR products were resolved on a 1.5% agarose gel, stained with ethidium bromide, and the intensity was quantified by Gel-Pro analysis software (ImageJ software, version 1.49n; National Institutes of Health, Bethesda, MD, USA). qPCR amplification was conducted using SYBR-Green q-RT-PCR kit reagents (Fermentas) according to the manufacturer's instructions.

    Modification:

    Article Title: G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV
    Article Snippet: Viral genome extraction and quantification BCBL-1 treated with either PhenDC3 or DMSO were collected by centrifugation (∼2 × 106 cells per sample) and washed twice with PBS before extracting the total DNA using a modified Hirt's lysis method ( ). .. The extracted total DNA was resuspended in 50 μl sterile water, and a 5 μl aliquot of the DNA was used for the qPCR amplification of the KSHV-ORF73-specific sequence on an ABI StepOnePlusTM Real-Time PCR machine (Applied Biosystems, Grand Island, NY, USA).

    Gel Purification:

    Article Title: The Characterization of RNA Viruses in Tropical Seawater Using Targeted PCR and Metagenomics
    Article Snippet: The qPCR amplification was performed on a 7300 real-time PCR system (Applied Biosystems) with Power SYBR green PCR Mastermix (Applied Biosystems). .. Standards consisted of 10-fold serial dilutions (3 × 101 to 3 × 109 molecules per reaction) of target molecule that had been cloned, amplified using appropriate primers, purified by agarose gel electrophoresis, and extracted with a MinElute Gel Purification kit (Qiagen).

    Sequencing:

    Article Title: The miR-99 family regulates the DNA damage response through its target SNF2H
    Article Snippet: .. For qPCR validation, Poly A tailing and cDNA preparation of mature microRNAs was performed using the NCODE miRNA amplification system (Invitrogen). qPCR amplification was performed using forward primers identical to the mature miRNA sequence, and NCODE universal reverse primers with Sybr Green ER (Invitrogen). .. Expression was normalized to u6snRNA, which used the primer sequence 5′-CTGCGCAAGGATGACACGCA-3′.

    Article Title: G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV
    Article Snippet: .. The extracted total DNA was resuspended in 50 μl sterile water, and a 5 μl aliquot of the DNA was used for the qPCR amplification of the KSHV-ORF73-specific sequence on an ABI StepOnePlusTM Real-Time PCR machine (Applied Biosystems, Grand Island, NY, USA). ..

    Concentration Assay:

    Article Title: The Characterization of RNA Viruses in Tropical Seawater Using Targeted PCR and Metagenomics
    Article Snippet: Reaction mixtures for cDNA synthesis (Superscript III; Invitrogen Corporation) consisted of 5 µl of the extracted, DNase-treated RNA template, a 0.2 mM concentration of each deoxynucleoside triphosphate, and a 0.5 µM concentration of reverse primer. .. The qPCR amplification was performed on a 7300 real-time PCR system (Applied Biosystems) with Power SYBR green PCR Mastermix (Applied Biosystems).

    Article Title: Roles of the Gac-Rsm pathway in the regulation of phenazine biosynthesis in Pseudomonas chlororaphis 30-84
    Article Snippet: Elimination of contaminating DNA was confirmed via qPCR amplification of the rpoD gene with SYBR green® dye on an ABI 9400HT PCR machine (Life Technologies, Carlsbad, CA). .. At the elution step, the two samples were pooled and concentrated using RiboMinus concentration modules (Life Technologies, Carlsbad, CA).

    Protease Inhibitor:

    Article Title: MOF-associated complexes ensure stem cell identity and Xist repression
    Article Snippet: Nuclei were resuspended in RIPA lysis buffer (1 × PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS + Roche Protease Inhibitor Cocktail Tablet, filtered through 0.2 micron filter unit). .. Purified ChIPed DNA was subjected to qPCR amplification (Applied Biosystems, Carlsbad, CA).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: S1P3 confers differential S1P migration by autoreactive and non-autoreactive immature B cells and is required for normal B cell development
    Article Snippet: RNA was isolated using TRIzol (InvitrogenLife Technologies), and trace amounts of DNA were removed usinga DNA-free kit (Ambion). cDNA was prepared from equivalent amounts of RNA using a SuperScript III First-Strand Synthesis Systemfor RT-PCR (Invitrogen Life Technologies). .. Quantitative PCR amplification was performed using Platinum SYBR Green qPCR SuperMix-UDG(Invitrogen Life Technologies) and detected on an MJ ResearchDNA Engine Opticon 2 real-time PCR machine.

    Article Title: Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells
    Article Snippet: .. RT-PCR products were resolved on a 1.5% agarose gel, stained with ethidium bromide, and the intensity was quantified by Gel-Pro analysis software (ImageJ software, version 1.49n; National Institutes of Health, Bethesda, MD, USA). qPCR amplification was conducted using SYBR-Green q-RT-PCR kit reagents (Fermentas) according to the manufacturer's instructions. ..

    Sonication:

    Article Title: Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model
    Article Snippet: DNA was then sheared by sonication, followed by immunoprecipitation with commercially available anti-acetylated histone H3 and H4 antibodies: H3K9ac, H4K5ac, and H4K12ac (Upstate). .. After reversal of cross-linking, quantitative PCR amplification of the resultant co-immunoprecipitated DNA was carried out with SYBR® Green in an ABI7900 sequencer (Applied Biosystems) using three sets of FXN primers (Pro, Up and Down) and mouse Gapdh primers, as previously described ( ).

    Quantitation Assay:

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment
    Article Snippet: Quantification of HIV total DNA and 2-LTR circular DNA QPCR quantitation of total HIV DNA in EcoHIV/NDK-infected mice and DNA standardization was described [ ]; HIV 2-LTR circular DNA was measured by QPCR as described [ ]. .. DNA from human cells was standardized by QPCR amplification of human β-globin QPCR (ThermoFisher Scientific) and custom-designed mouse β-globin QPCR [ ].

    Cellular Antioxidant Activity Assay:

    Article Title: Roles of the Gac-Rsm pathway in the regulation of phenazine biosynthesis in Pseudomonas chlororaphis 30-84
    Article Snippet: Cell cultures collected at OD600 = 1.2 were diluted to OD600 = 0.3 with AB + 2% CAA broth. .. Elimination of contaminating DNA was confirmed via qPCR amplification of the rpoD gene with SYBR green® dye on an ABI 9400HT PCR machine (Life Technologies, Carlsbad, CA).

    RNA Sequencing Assay:

    Article Title: Roles of the Gac-Rsm pathway in the regulation of phenazine biosynthesis in Pseudomonas chlororaphis 30-84
    Article Snippet: Paragraph title: RNA preparation for RNA-seq and qPCR analyses ... Elimination of contaminating DNA was confirmed via qPCR amplification of the rpoD gene with SYBR green® dye on an ABI 9400HT PCR machine (Life Technologies, Carlsbad, CA).

    Isolation:

    Article Title: The miR-99 family regulates the DNA damage response through its target SNF2H
    Article Snippet: miRNA expression analysis RNA was isolated from cells using Trizol extraction (Invitrogen). .. For qPCR validation, Poly A tailing and cDNA preparation of mature microRNAs was performed using the NCODE miRNA amplification system (Invitrogen). qPCR amplification was performed using forward primers identical to the mature miRNA sequence, and NCODE universal reverse primers with Sybr Green ER (Invitrogen).

    Article Title: Antiviral therapy may decrease HBx, affecting cccDNA and MSL2 in hepatocarcinogenesis
    Article Snippet: Intrahepatic levels of cccDNA in tumor and peri-tumor tissues were compared. cccDNA was isolated by digestion from 300 mg of total DNA with Plasmid-safe™ ATP-dependent DNase and diluted with 20 ml of diethyl pyrocarbonate water. .. A total of 1 ml of cccDNA was then used for qPCR amplification (7500 Real-time PCR Instrument system; Applied Biosystems; Thermo Fisher Scientific, Inc.), which was performed using TOPreal™ qPCR PreMIX SYBR Green (Enzynomics). β-actin amplicons were used as the internal reference for subsequent PCR analysis.

    Article Title: S1P3 confers differential S1P migration by autoreactive and non-autoreactive immature B cells and is required for normal B cell development
    Article Snippet: RNA was isolated using TRIzol (InvitrogenLife Technologies), and trace amounts of DNA were removed usinga DNA-free kit (Ambion). cDNA was prepared from equivalent amounts of RNA using a SuperScript III First-Strand Synthesis Systemfor RT-PCR (Invitrogen Life Technologies). .. Quantitative PCR amplification was performed using Platinum SYBR Green qPCR SuperMix-UDG(Invitrogen Life Technologies) and detected on an MJ ResearchDNA Engine Opticon 2 real-time PCR machine.

    Article Title: miR‐515‐5p controls cancer cell migration through MARK4 regulation
    Article Snippet: MicroRNAs and mRNA expression RNA was isolated using TRIzol reagent according to the manufacturer instructions. .. To analyse mRNA expression, both cDNA conversion and qPCR amplification were performed as recommended by Applied Biosystems using the High‐Capacity cDNA Reverse Transcription Kit and Fast SYBR® Green Master Mix, respectively.

    Article Title: Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells
    Article Snippet: Paragraph title: RNA isolation, RT-PCR and qPCR ... RT-PCR products were resolved on a 1.5% agarose gel, stained with ethidium bromide, and the intensity was quantified by Gel-Pro analysis software (ImageJ software, version 1.49n; National Institutes of Health, Bethesda, MD, USA). qPCR amplification was conducted using SYBR-Green q-RT-PCR kit reagents (Fermentas) according to the manufacturer's instructions.

    Size-exclusion Chromatography:

    Article Title: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus
    Article Snippet: RC21 and RC1 were replaced by Tag-ZK21 primer (5′-ggccgtcatggtggcgaataaCCTGACAACACTAAaATTGGTGC-3′) and Tag-ZK1 primer (5′-ggccgtcatggtggcgaataaAGGATCATAGGTGATGAAGAAAAGT-3′). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers’ instructions. qPCR amplification was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), in a PikoReal 96 Real-Time PCR system (ThermoFisher Scientific). .. The cycle conditions were uracil-DNA glycosylase (UDG) activation at 50 °C for 2 min, dual-lock DNA polymerase at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing at 55 °C for 15 sec, and extension at 72 °C for 1 min. An MR766 infectious clone [ ] was used to generate a standard curve, and was subject to the same strand specific RT-qPCR protocol.

    Purification:

    Article Title: The miR-99 family regulates the DNA damage response through its target SNF2H
    Article Snippet: For microarray analysis, RNA was further purified using RNAeasy RNA cleanup kit (Qiagen). .. For qPCR validation, Poly A tailing and cDNA preparation of mature microRNAs was performed using the NCODE miRNA amplification system (Invitrogen). qPCR amplification was performed using forward primers identical to the mature miRNA sequence, and NCODE universal reverse primers with Sybr Green ER (Invitrogen).

    Article Title: The Characterization of RNA Viruses in Tropical Seawater Using Targeted PCR and Metagenomics
    Article Snippet: The qPCR amplification was performed on a 7300 real-time PCR system (Applied Biosystems) with Power SYBR green PCR Mastermix (Applied Biosystems). .. Standards consisted of 10-fold serial dilutions (3 × 101 to 3 × 109 molecules per reaction) of target molecule that had been cloned, amplified using appropriate primers, purified by agarose gel electrophoresis, and extracted with a MinElute Gel Purification kit (Qiagen).

    Article Title: PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein
    Article Snippet: Quantitative real-time PCR (RT–qPCR) Total RNA was extracted using the TRIZOL reagent (Invitrogen) and further purified with the PureLink RNA Mini Kit (Invitrogen). .. A 200-ng sample of total RNA was reverse-transcribed with the Superscript III Reverse Transcriptase (Invitrogen), using 10 μ random hexamer primers (Roche) for the Superscript Reverse Transcriptase (Invitrogen), according to the manufacturer's instructions. qPCR amplification was performed with a 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA) using the Platinum SYBR Green qPCR Supermix-UDG with ROX (Invitrogen), using specific primers (200 n) as listed in .

    Article Title: MOF-associated complexes ensure stem cell identity and Xist repression
    Article Snippet: .. Purified ChIPed DNA was subjected to qPCR amplification (Applied Biosystems, Carlsbad, CA). ..

    Polymerase Chain Reaction:

    Article Title: The Characterization of RNA Viruses in Tropical Seawater Using Targeted PCR and Metagenomics
    Article Snippet: .. The qPCR amplification was performed on a 7300 real-time PCR system (Applied Biosystems) with Power SYBR green PCR Mastermix (Applied Biosystems). .. The reaction mixtures contained 12.5 µl SYBR green PCR master mix (Applied Biosystems), a 0.2 µM concentration of each primer, and 2 µl of sample cDNA template, with a final volume of 25 µl.

    Article Title: Antiviral therapy may decrease HBx, affecting cccDNA and MSL2 in hepatocarcinogenesis
    Article Snippet: .. A total of 1 ml of cccDNA was then used for qPCR amplification (7500 Real-time PCR Instrument system; Applied Biosystems; Thermo Fisher Scientific, Inc.), which was performed using TOPreal™ qPCR PreMIX SYBR Green (Enzynomics). β-actin amplicons were used as the internal reference for subsequent PCR analysis. .. β-actin amplicons were used as the internal reference for subsequent PCR analysis.

    Article Title: Insect Neuropeptide Bursicon Homodimers Induce Innate Immune and Stress Genes during Molting by Activating the NF-?B Transcription Factor Relish
    Article Snippet: Gene specific primers ( ) were used for qPCR amplification of AMP genes. qPCR amplification and analysis were carried out on an Applied Biosystems (ABI) 7500 Fast Real-Time PCR System. .. The PCR program was: hold at 95°C for 10 min and then at 95°C for 15 seconds and 60°C for 1 min, repeating 40 cycles.

    Article Title: G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV
    Article Snippet: The PCR primers used for the KSHV genome quantification were selected from the ORF73 gene, as previously described ( , ). .. The extracted total DNA was resuspended in 50 μl sterile water, and a 5 μl aliquot of the DNA was used for the qPCR amplification of the KSHV-ORF73-specific sequence on an ABI StepOnePlusTM Real-Time PCR machine (Applied Biosystems, Grand Island, NY, USA).

    Article Title: miR‐515‐5p controls cancer cell migration through MARK4 regulation
    Article Snippet: MicroRNA levels were quantified using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems), TaqMan 2× Universal PCR Master Mix No AmpErase UNG (Applied Biosystems) and has‐pre‐miR‐515‐5p TaqMan primers (Applied Biosystems). .. To analyse mRNA expression, both cDNA conversion and qPCR amplification were performed as recommended by Applied Biosystems using the High‐Capacity cDNA Reverse Transcription Kit and Fast SYBR® Green Master Mix, respectively.

    Article Title: Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells
    Article Snippet: The reaction conditions were established by 12.5 µl 2X PCR Master (Tiangen Biotech, Beijing, China), 3 µl cDNA template, and 0.5 µl of each primer. .. RT-PCR products were resolved on a 1.5% agarose gel, stained with ethidium bromide, and the intensity was quantified by Gel-Pro analysis software (ImageJ software, version 1.49n; National Institutes of Health, Bethesda, MD, USA). qPCR amplification was conducted using SYBR-Green q-RT-PCR kit reagents (Fermentas) according to the manufacturer's instructions.

    Article Title: MOF-associated complexes ensure stem cell identity and Xist repression
    Article Snippet: Washed beads were resuspended in 100 μl of IP elution buffer and subjected to overnight reverse cross-linking (RNase and proteinase K digestions) followed by DNA purification (DNA was purified using Minelute PCR purification kit from Qiagen, Germany). .. Purified ChIPed DNA was subjected to qPCR amplification (Applied Biosystems, Carlsbad, CA).

    Article Title: Roles of the Gac-Rsm pathway in the regulation of phenazine biosynthesis in Pseudomonas chlororaphis 30-84
    Article Snippet: .. Elimination of contaminating DNA was confirmed via qPCR amplification of the rpoD gene with SYBR green® dye on an ABI 9400HT PCR machine (Life Technologies, Carlsbad, CA). ..

    FACS:

    Article Title: S1P3 confers differential S1P migration by autoreactive and non-autoreactive immature B cells and is required for normal B cell development
    Article Snippet: Paragraph title: Cell sorting and quantitative PCR ... Quantitative PCR amplification was performed using Platinum SYBR Green qPCR SuperMix-UDG(Invitrogen Life Technologies) and detected on an MJ ResearchDNA Engine Opticon 2 real-time PCR machine.

    Mouse Assay:

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment
    Article Snippet: Quantification of HIV total DNA and 2-LTR circular DNA QPCR quantitation of total HIV DNA in EcoHIV/NDK-infected mice and DNA standardization was described [ ]; HIV 2-LTR circular DNA was measured by QPCR as described [ ]. .. DNA from human cells was standardized by QPCR amplification of human β-globin QPCR (ThermoFisher Scientific) and custom-designed mouse β-globin QPCR [ ].

    Article Title: S1P3 confers differential S1P migration by autoreactive and non-autoreactive immature B cells and is required for normal B cell development
    Article Snippet: Bone marrow cells were isolated from 3-83Ig, H-2d or 3-83Ig, Rag1−/− , H-2b mice and sorted as B220+ IgD− CD23− CD43− using a MoFlow sorter. .. Quantitative PCR amplification was performed using Platinum SYBR Green qPCR SuperMix-UDG(Invitrogen Life Technologies) and detected on an MJ ResearchDNA Engine Opticon 2 real-time PCR machine.

    Chromatin Immunoprecipitation:

    Article Title: Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model
    Article Snippet: Paragraph title: ChIP analysis ... After reversal of cross-linking, quantitative PCR amplification of the resultant co-immunoprecipitated DNA was carried out with SYBR® Green in an ABI7900 sequencer (Applied Biosystems) using three sets of FXN primers (Pro, Up and Down) and mouse Gapdh primers, as previously described ( ).

    Article Title: MOF-associated complexes ensure stem cell identity and Xist repression
    Article Snippet: Paragraph title: Immunoprecipitation assays (IP and ChIP) ... Purified ChIPed DNA was subjected to qPCR amplification (Applied Biosystems, Carlsbad, CA).

    Plasmid Preparation:

    Article Title: Antiviral therapy may decrease HBx, affecting cccDNA and MSL2 in hepatocarcinogenesis
    Article Snippet: Intrahepatic levels of cccDNA in tumor and peri-tumor tissues were compared. cccDNA was isolated by digestion from 300 mg of total DNA with Plasmid-safe™ ATP-dependent DNase and diluted with 20 ml of diethyl pyrocarbonate water. .. A total of 1 ml of cccDNA was then used for qPCR amplification (7500 Real-time PCR Instrument system; Applied Biosystems; Thermo Fisher Scientific, Inc.), which was performed using TOPreal™ qPCR PreMIX SYBR Green (Enzynomics). β-actin amplicons were used as the internal reference for subsequent PCR analysis.

    Article Title: G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV
    Article Snippet: Two-fold serial dilutions of the pA3F-LANA plasmid were used as template in qPCR reactions to produce a standard curve for the quantifications. .. The extracted total DNA was resuspended in 50 μl sterile water, and a 5 μl aliquot of the DNA was used for the qPCR amplification of the KSHV-ORF73-specific sequence on an ABI StepOnePlusTM Real-Time PCR machine (Applied Biosystems, Grand Island, NY, USA).

    Software:

    Article Title: Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells
    Article Snippet: .. RT-PCR products were resolved on a 1.5% agarose gel, stained with ethidium bromide, and the intensity was quantified by Gel-Pro analysis software (ImageJ software, version 1.49n; National Institutes of Health, Bethesda, MD, USA). qPCR amplification was conducted using SYBR-Green q-RT-PCR kit reagents (Fermentas) according to the manufacturer's instructions. ..

    Real-time Polymerase Chain Reaction:

    Article Title: The miR-99 family regulates the DNA damage response through its target SNF2H
    Article Snippet: .. For qPCR validation, Poly A tailing and cDNA preparation of mature microRNAs was performed using the NCODE miRNA amplification system (Invitrogen). qPCR amplification was performed using forward primers identical to the mature miRNA sequence, and NCODE universal reverse primers with Sybr Green ER (Invitrogen). .. Expression was normalized to u6snRNA, which used the primer sequence 5′-CTGCGCAAGGATGACACGCA-3′.

    Article Title: The Characterization of RNA Viruses in Tropical Seawater Using Targeted PCR and Metagenomics
    Article Snippet: .. The qPCR amplification was performed on a 7300 real-time PCR system (Applied Biosystems) with Power SYBR green PCR Mastermix (Applied Biosystems). .. The reaction mixtures contained 12.5 µl SYBR green PCR master mix (Applied Biosystems), a 0.2 µM concentration of each primer, and 2 µl of sample cDNA template, with a final volume of 25 µl.

    Article Title: Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model
    Article Snippet: .. After reversal of cross-linking, quantitative PCR amplification of the resultant co-immunoprecipitated DNA was carried out with SYBR® Green in an ABI7900 sequencer (Applied Biosystems) using three sets of FXN primers (Pro, Up and Down) and mouse Gapdh primers, as previously described ( ). .. Each tissue sample was subjected to two independent ChIP procedures, followed by triplicate quantitative PCR analysis.

    Article Title: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus
    Article Snippet: .. RC21 and RC1 were replaced by Tag-ZK21 primer (5′-ggccgtcatggtggcgaataaCCTGACAACACTAAaATTGGTGC-3′) and Tag-ZK1 primer (5′-ggccgtcatggtggcgaataaAGGATCATAGGTGATGAAGAAAAGT-3′). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers’ instructions. qPCR amplification was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), in a PikoReal 96 Real-Time PCR system (ThermoFisher Scientific). .. The cycle conditions were uracil-DNA glycosylase (UDG) activation at 50 °C for 2 min, dual-lock DNA polymerase at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing at 55 °C for 15 sec, and extension at 72 °C for 1 min. An MR766 infectious clone [ ] was used to generate a standard curve, and was subject to the same strand specific RT-qPCR protocol.

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment
    Article Snippet: .. DNA from human cells was standardized by QPCR amplification of human β-globin QPCR (ThermoFisher Scientific) and custom-designed mouse β-globin QPCR [ ]. ..

    Article Title: Antiviral therapy may decrease HBx, affecting cccDNA and MSL2 in hepatocarcinogenesis
    Article Snippet: .. A total of 1 ml of cccDNA was then used for qPCR amplification (7500 Real-time PCR Instrument system; Applied Biosystems; Thermo Fisher Scientific, Inc.), which was performed using TOPreal™ qPCR PreMIX SYBR Green (Enzynomics). β-actin amplicons were used as the internal reference for subsequent PCR analysis. .. β-actin amplicons were used as the internal reference for subsequent PCR analysis.

    Article Title: Insect Neuropeptide Bursicon Homodimers Induce Innate Immune and Stress Genes during Molting by Activating the NF-?B Transcription Factor Relish
    Article Snippet: .. Gene specific primers ( ) were used for qPCR amplification of AMP genes. qPCR amplification and analysis were carried out on an Applied Biosystems (ABI) 7500 Fast Real-Time PCR System. .. The final reaction volume was 25 µl using ABI SYBR green Supermix (ABI).

    Article Title: G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV
    Article Snippet: .. The extracted total DNA was resuspended in 50 μl sterile water, and a 5 μl aliquot of the DNA was used for the qPCR amplification of the KSHV-ORF73-specific sequence on an ABI StepOnePlusTM Real-Time PCR machine (Applied Biosystems, Grand Island, NY, USA). ..

    Article Title: S1P3 confers differential S1P migration by autoreactive and non-autoreactive immature B cells and is required for normal B cell development
    Article Snippet: .. Quantitative PCR amplification was performed using Platinum SYBR Green qPCR SuperMix-UDG(Invitrogen Life Technologies) and detected on an MJ ResearchDNA Engine Opticon 2 real-time PCR machine. ..

    Article Title: miR‐515‐5p controls cancer cell migration through MARK4 regulation
    Article Snippet: .. To analyse mRNA expression, both cDNA conversion and qPCR amplification were performed as recommended by Applied Biosystems using the High‐Capacity cDNA Reverse Transcription Kit and Fast SYBR® Green Master Mix, respectively. ..

    Article Title: PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein
    Article Snippet: .. A 200-ng sample of total RNA was reverse-transcribed with the Superscript III Reverse Transcriptase (Invitrogen), using 10 μ random hexamer primers (Roche) for the Superscript Reverse Transcriptase (Invitrogen), according to the manufacturer's instructions. qPCR amplification was performed with a 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA) using the Platinum SYBR Green qPCR Supermix-UDG with ROX (Invitrogen), using specific primers (200 n) as listed in . .. In each experiment, the housekeeping genes, PPIB and YWHAZ , were amplified as a reference standard for normalization.

    Article Title: A Reverse Time-Course Method for Transcriptional Chase Analyses of mRNA Half-Lives in Cultured Cells
    Article Snippet: .. First-strand cDNA was subjected to qPCR amplification using Taqman assays specific for β-globin (#00747223_g1) or endogenous control β-actin (#99999903_m1) mRNAs, according to the manufacturer’s recommendations (Applied Biosystems). .. Samples were analyzed using an ABI 7500 Real-Time PCR system (Applied Biosystems).

    Article Title: Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells
    Article Snippet: .. RT-PCR products were resolved on a 1.5% agarose gel, stained with ethidium bromide, and the intensity was quantified by Gel-Pro analysis software (ImageJ software, version 1.49n; National Institutes of Health, Bethesda, MD, USA). qPCR amplification was conducted using SYBR-Green q-RT-PCR kit reagents (Fermentas) according to the manufacturer's instructions. ..

    Article Title: MOF-associated complexes ensure stem cell identity and Xist repression
    Article Snippet: .. Purified ChIPed DNA was subjected to qPCR amplification (Applied Biosystems, Carlsbad, CA). ..

    Article Title: Roles of the Gac-Rsm pathway in the regulation of phenazine biosynthesis in Pseudomonas chlororaphis 30-84
    Article Snippet: .. Elimination of contaminating DNA was confirmed via qPCR amplification of the rpoD gene with SYBR green® dye on an ABI 9400HT PCR machine (Life Technologies, Carlsbad, CA). ..

    RNA Extraction:

    Article Title: Roles of the Gac-Rsm pathway in the regulation of phenazine biosynthesis in Pseudomonas chlororaphis 30-84
    Article Snippet: RNA extraction was performed as described previously (Wang et al. ) with one exception: contaminating genomic DNA was removed off-column with Turbo DNA-free DNAse (Life Technologies, Carlsbad, CA). .. Elimination of contaminating DNA was confirmed via qPCR amplification of the rpoD gene with SYBR green® dye on an ABI 9400HT PCR machine (Life Technologies, Carlsbad, CA).

    Agarose Gel Electrophoresis:

    Article Title: The Characterization of RNA Viruses in Tropical Seawater Using Targeted PCR and Metagenomics
    Article Snippet: The qPCR amplification was performed on a 7300 real-time PCR system (Applied Biosystems) with Power SYBR green PCR Mastermix (Applied Biosystems). .. Standards consisted of 10-fold serial dilutions (3 × 101 to 3 × 109 molecules per reaction) of target molecule that had been cloned, amplified using appropriate primers, purified by agarose gel electrophoresis, and extracted with a MinElute Gel Purification kit (Qiagen).

    Article Title: Insect Neuropeptide Bursicon Homodimers Induce Innate Immune and Stress Genes during Molting by Activating the NF-?B Transcription Factor Relish
    Article Snippet: Gene specific primers ( ) were used for qPCR amplification of AMP genes. qPCR amplification and analysis were carried out on an Applied Biosystems (ABI) 7500 Fast Real-Time PCR System. .. The specificity of the SYBR green PCR signal was further confirmed by a melting curve analysis and agarose gel electrophoresis.

    Article Title: Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells
    Article Snippet: .. RT-PCR products were resolved on a 1.5% agarose gel, stained with ethidium bromide, and the intensity was quantified by Gel-Pro analysis software (ImageJ software, version 1.49n; National Institutes of Health, Bethesda, MD, USA). qPCR amplification was conducted using SYBR-Green q-RT-PCR kit reagents (Fermentas) according to the manufacturer's instructions. ..

    In Vitro:

    Article Title: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus
    Article Snippet: In vitro transcribed RNA from the HCV infectious clone was used to generate a standard curve. .. RC21 and RC1 were replaced by Tag-ZK21 primer (5′-ggccgtcatggtggcgaataaCCTGACAACACTAAaATTGGTGC-3′) and Tag-ZK1 primer (5′-ggccgtcatggtggcgaataaAGGATCATAGGTGATGAAGAAAAGT-3′). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers’ instructions. qPCR amplification was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), in a PikoReal 96 Real-Time PCR system (ThermoFisher Scientific).

    Random Hexamer Labeling:

    Article Title: PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein
    Article Snippet: .. A 200-ng sample of total RNA was reverse-transcribed with the Superscript III Reverse Transcriptase (Invitrogen), using 10 μ random hexamer primers (Roche) for the Superscript Reverse Transcriptase (Invitrogen), according to the manufacturer's instructions. qPCR amplification was performed with a 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA) using the Platinum SYBR Green qPCR Supermix-UDG with ROX (Invitrogen), using specific primers (200 n) as listed in . .. In each experiment, the housekeeping genes, PPIB and YWHAZ , were amplified as a reference standard for normalization.

    Activation Assay:

    Article Title: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus
    Article Snippet: RC21 and RC1 were replaced by Tag-ZK21 primer (5′-ggccgtcatggtggcgaataaCCTGACAACACTAAaATTGGTGC-3′) and Tag-ZK1 primer (5′-ggccgtcatggtggcgaataaAGGATCATAGGTGATGAAGAAAAGT-3′). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers’ instructions. qPCR amplification was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), in a PikoReal 96 Real-Time PCR system (ThermoFisher Scientific). .. The cycle conditions were uracil-DNA glycosylase (UDG) activation at 50 °C for 2 min, dual-lock DNA polymerase at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing at 55 °C for 15 sec, and extension at 72 °C for 1 min. An MR766 infectious clone [ ] was used to generate a standard curve, and was subject to the same strand specific RT-qPCR protocol.

    Immunoprecipitation:

    Article Title: Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model
    Article Snippet: For each experiment, normal rabbit serum (SIGMA) was used as a minus antibody immunoprecipitation control. .. After reversal of cross-linking, quantitative PCR amplification of the resultant co-immunoprecipitated DNA was carried out with SYBR® Green in an ABI7900 sequencer (Applied Biosystems) using three sets of FXN primers (Pro, Up and Down) and mouse Gapdh primers, as previously described ( ).

    Article Title: MOF-associated complexes ensure stem cell identity and Xist repression
    Article Snippet: Paragraph title: Immunoprecipitation assays (IP and ChIP) ... Purified ChIPed DNA was subjected to qPCR amplification (Applied Biosystems, Carlsbad, CA).

    DNA Purification:

    Article Title: MOF-associated complexes ensure stem cell identity and Xist repression
    Article Snippet: Washed beads were resuspended in 100 μl of IP elution buffer and subjected to overnight reverse cross-linking (RNase and proteinase K digestions) followed by DNA purification (DNA was purified using Minelute PCR purification kit from Qiagen, Germany). .. Purified ChIPed DNA was subjected to qPCR amplification (Applied Biosystems, Carlsbad, CA).

    Lysis:

    Article Title: G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV
    Article Snippet: Viral genome extraction and quantification BCBL-1 treated with either PhenDC3 or DMSO were collected by centrifugation (∼2 × 106 cells per sample) and washed twice with PBS before extracting the total DNA using a modified Hirt's lysis method ( ). .. The extracted total DNA was resuspended in 50 μl sterile water, and a 5 μl aliquot of the DNA was used for the qPCR amplification of the KSHV-ORF73-specific sequence on an ABI StepOnePlusTM Real-Time PCR machine (Applied Biosystems, Grand Island, NY, USA).

    Article Title: MOF-associated complexes ensure stem cell identity and Xist repression
    Article Snippet: After incubation, 50 μl of 50% slurry bead solution was added for another incubation period (2 hr), then beads were washed: four times for 15 min with RIPA lysis buffer, two times for 1 min with LiCl IP wash buffer (250 mM LiCl, 10 mM Tris–HCl pH 8.0, 1 mM EDTA, 0.5% NP-40, 0.5% DOC, filtered through 0.2 micron filter unit), two times for 1 min with TE buffer (1 mM Tris–HCl pH 8.0, 1 mM EDTA, filtered through 0.2 micron filter unit). .. Purified ChIPed DNA was subjected to qPCR amplification (Applied Biosystems, Carlsbad, CA).

    Staining:

    Article Title: Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells
    Article Snippet: .. RT-PCR products were resolved on a 1.5% agarose gel, stained with ethidium bromide, and the intensity was quantified by Gel-Pro analysis software (ImageJ software, version 1.49n; National Institutes of Health, Bethesda, MD, USA). qPCR amplification was conducted using SYBR-Green q-RT-PCR kit reagents (Fermentas) according to the manufacturer's instructions. ..

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    Thermo Fisher qpcr amplification
    Both EcoHIV and MLV infect mouse brain but only EcoHIV-infected mice develop cognitive impairment. A. Design of the experiment. B. Groups of mice were gavaged with cART or vehicle before and during infection with indicated viruses or vehicle. At day 21 after infection, mice were tested for errors (left panel) and latency (middle panel) in finding the hidden platform, the right panel shows latency to reach the visible platform. RT represents the retention trial. C. Two days after completion of RAWM, the same mice were used to test contextual response to fear conditioning training. The percentage of freezing time was calculated for contextual fear memory deficit in each group of mice. D. Mice were euthanized on day 40 after infection and levels of total vDNA in spleen and brain measured by <t>QPCR</t> (left panel) and integrated viral <t>DNA</t> measured by nested QPCR in PM (right panel). All data are mean ± standard errors. * P
    Qpcr Amplification, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 257 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Both EcoHIV and MLV infect mouse brain but only EcoHIV-infected mice develop cognitive impairment. A. Design of the experiment. B. Groups of mice were gavaged with cART or vehicle before and during infection with indicated viruses or vehicle. At day 21 after infection, mice were tested for errors (left panel) and latency (middle panel) in finding the hidden platform, the right panel shows latency to reach the visible platform. RT represents the retention trial. C. Two days after completion of RAWM, the same mice were used to test contextual response to fear conditioning training. The percentage of freezing time was calculated for contextual fear memory deficit in each group of mice. D. Mice were euthanized on day 40 after infection and levels of total vDNA in spleen and brain measured by QPCR (left panel) and integrated viral DNA measured by nested QPCR in PM (right panel). All data are mean ± standard errors. * P

    Journal: PLoS Pathogens

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

    doi: 10.1371/journal.ppat.1007061

    Figure Lengend Snippet: Both EcoHIV and MLV infect mouse brain but only EcoHIV-infected mice develop cognitive impairment. A. Design of the experiment. B. Groups of mice were gavaged with cART or vehicle before and during infection with indicated viruses or vehicle. At day 21 after infection, mice were tested for errors (left panel) and latency (middle panel) in finding the hidden platform, the right panel shows latency to reach the visible platform. RT represents the retention trial. C. Two days after completion of RAWM, the same mice were used to test contextual response to fear conditioning training. The percentage of freezing time was calculated for contextual fear memory deficit in each group of mice. D. Mice were euthanized on day 40 after infection and levels of total vDNA in spleen and brain measured by QPCR (left panel) and integrated viral DNA measured by nested QPCR in PM (right panel). All data are mean ± standard errors. * P

    Article Snippet: DNA from human cells was standardized by QPCR amplification of human β-globin QPCR (ThermoFisher Scientific) and custom-designed mouse β-globin QPCR [ ].

    Techniques: Infection, Mouse Assay, Real-time Polymerase Chain Reaction

    EcoHIV establishes latent reservoirs in resting CD4 + cells that are inducible by epigenetic modulators. A. CD4 + T cells isolated from spleen at day 25 post-infection were cocultured with uninfected cells in the presence or absence of ABC as indicated prior to vDNA amplification, each symbol represents culture from a single mouse. B. Six weeks after EcoHIV infection, CD4 + splenic T cells were harvested and purified from donor male 129X1/SvJ mice and injected into the recipient female 129X1 nude mice; their PM were collected after one week. Y chromosome and EcoHIV gag RNA levels in PM were determined by QPCR. Each symbol represents a single mouse. C.-D. Twelve weeks after infection, resting CD4 + T cells were purified from the spleen and C. The levels of integrated EcoHIV DNA and D. Genomic RNA were measured by QPCR. E. Eight weeks after EcoHIV infection of mice, resting CD4 + T cells were isolated and exposed to the agents indicated in culture for 2 days prior to collection and measurement of EcoHIV mRNA QPCR. F. Six weeks after infection by EcoHIV-Luc, mice were treated as shown, euthanized and splenic CD4 + cells isolated for measurement of virus RNA expression (left panel) or protein expression (right panel). * P

    Journal: PLoS Pathogens

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

    doi: 10.1371/journal.ppat.1007061

    Figure Lengend Snippet: EcoHIV establishes latent reservoirs in resting CD4 + cells that are inducible by epigenetic modulators. A. CD4 + T cells isolated from spleen at day 25 post-infection were cocultured with uninfected cells in the presence or absence of ABC as indicated prior to vDNA amplification, each symbol represents culture from a single mouse. B. Six weeks after EcoHIV infection, CD4 + splenic T cells were harvested and purified from donor male 129X1/SvJ mice and injected into the recipient female 129X1 nude mice; their PM were collected after one week. Y chromosome and EcoHIV gag RNA levels in PM were determined by QPCR. Each symbol represents a single mouse. C.-D. Twelve weeks after infection, resting CD4 + T cells were purified from the spleen and C. The levels of integrated EcoHIV DNA and D. Genomic RNA were measured by QPCR. E. Eight weeks after EcoHIV infection of mice, resting CD4 + T cells were isolated and exposed to the agents indicated in culture for 2 days prior to collection and measurement of EcoHIV mRNA QPCR. F. Six weeks after infection by EcoHIV-Luc, mice were treated as shown, euthanized and splenic CD4 + cells isolated for measurement of virus RNA expression (left panel) or protein expression (right panel). * P

    Article Snippet: DNA from human cells was standardized by QPCR amplification of human β-globin QPCR (ThermoFisher Scientific) and custom-designed mouse β-globin QPCR [ ].

    Techniques: Isolation, Infection, Amplification, Purification, Mouse Assay, Injection, Real-time Polymerase Chain Reaction, RNA Expression, Expressing

    Mouse macrophages are susceptible to EcoHIV but not MLV. A. Expression of CAT-1 was determined in peritoneal cells (PC) and thymus (TH) of C57BL/6 mice by QPCR. B. Ten days after EcoHIV or MLV infection of mice, the indicated tissues were harvested for measurement of viral nucleic acids by QPCR. C. Eight weeks after mouse infection by EcoHIV or MLV, F4/80 + macrophages were purified from PC and integrated viral DNA in unfractionated or F4/80 + PC was measured by nested-QPCR. D. C57BL/6 euthymic and athymic mice were infected by EcoHIV and PC harvested from groups at the times indicated to measure spliced EcoHIV RNA. Mean and standard errors are shown (* P

    Journal: PLoS Pathogens

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

    doi: 10.1371/journal.ppat.1007061

    Figure Lengend Snippet: Mouse macrophages are susceptible to EcoHIV but not MLV. A. Expression of CAT-1 was determined in peritoneal cells (PC) and thymus (TH) of C57BL/6 mice by QPCR. B. Ten days after EcoHIV or MLV infection of mice, the indicated tissues were harvested for measurement of viral nucleic acids by QPCR. C. Eight weeks after mouse infection by EcoHIV or MLV, F4/80 + macrophages were purified from PC and integrated viral DNA in unfractionated or F4/80 + PC was measured by nested-QPCR. D. C57BL/6 euthymic and athymic mice were infected by EcoHIV and PC harvested from groups at the times indicated to measure spliced EcoHIV RNA. Mean and standard errors are shown (* P

    Article Snippet: DNA from human cells was standardized by QPCR amplification of human β-globin QPCR (ThermoFisher Scientific) and custom-designed mouse β-globin QPCR [ ].

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

    EcoHIV infects mice and induces antiviral immune responses but not immunodeficiency in mice. A.-B. Kinetics of production of total EcoHIV DNA (left panels), integrated EcoHIV DNA (middle panels), and genomic EcoHIV RNA (right panels) were measured by QPCR in A. spleen and B. PC at the times indicated after infection. Each point represents a single mouse. C. EcoHIV gag RNA burden at each time point in copies per ml of whole blood was measured at the times after infection indicated. The dashed line indicates limit of detection of 10 copies. D. The frequency of interferon-γ expression by CD4 + or CD8 + spleen cells at the time indicated after EcoHIV or mock infection was measured by flow cytometry. E. The number of CD4 + and CD8 + T cells was measured by flow cytometry at the indicated times and the CD4 + :CD8 + ratio is shown. Symbols represent individual mice, the horizontal lines represent the mean. F. Longitudinal anti-NDK Gag antibodies in 3 EcoHIV-infected mice were measured by ELISA at the times indicated after infection. Each panel represents titers in a single mouse, mean +/- standard errors are shown.

    Journal: PLoS Pathogens

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

    doi: 10.1371/journal.ppat.1007061

    Figure Lengend Snippet: EcoHIV infects mice and induces antiviral immune responses but not immunodeficiency in mice. A.-B. Kinetics of production of total EcoHIV DNA (left panels), integrated EcoHIV DNA (middle panels), and genomic EcoHIV RNA (right panels) were measured by QPCR in A. spleen and B. PC at the times indicated after infection. Each point represents a single mouse. C. EcoHIV gag RNA burden at each time point in copies per ml of whole blood was measured at the times after infection indicated. The dashed line indicates limit of detection of 10 copies. D. The frequency of interferon-γ expression by CD4 + or CD8 + spleen cells at the time indicated after EcoHIV or mock infection was measured by flow cytometry. E. The number of CD4 + and CD8 + T cells was measured by flow cytometry at the indicated times and the CD4 + :CD8 + ratio is shown. Symbols represent individual mice, the horizontal lines represent the mean. F. Longitudinal anti-NDK Gag antibodies in 3 EcoHIV-infected mice were measured by ELISA at the times indicated after infection. Each panel represents titers in a single mouse, mean +/- standard errors are shown.

    Article Snippet: DNA from human cells was standardized by QPCR amplification of human β-globin QPCR (ThermoFisher Scientific) and custom-designed mouse β-globin QPCR [ ].

    Techniques: Mouse Assay, Real-time Polymerase Chain Reaction, Infection, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    EcoHIV integrates efficiently in the murine host genome. A. Schematic representation of strategy for assay of integration. In the first round PCR, primers, B1-F and B1-R, from the host B1 repetitive elements and a HIV-specific primer, Fnef, were used to amplify a region of integrated virus and the host flanking region. The second nested QPCR based on the HIV RU5 region was performed using the preamplified products. B. Integration standard curve was generated by logarithmic regression of the amplified IS sample signals. C.-F. Mice (n = 4/system) were pretreated with ABC or RAL 24 h before EcoHIV infection, euthanized 3 days later, and spleens collected for measurement of C. total, D. integrated, and E. 2-LTR circle vDNA; F. spliced vRNA by QPCR of DNA or cDNA as described in Methods.

    Journal: PLoS Pathogens

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

    doi: 10.1371/journal.ppat.1007061

    Figure Lengend Snippet: EcoHIV integrates efficiently in the murine host genome. A. Schematic representation of strategy for assay of integration. In the first round PCR, primers, B1-F and B1-R, from the host B1 repetitive elements and a HIV-specific primer, Fnef, were used to amplify a region of integrated virus and the host flanking region. The second nested QPCR based on the HIV RU5 region was performed using the preamplified products. B. Integration standard curve was generated by logarithmic regression of the amplified IS sample signals. C.-F. Mice (n = 4/system) were pretreated with ABC or RAL 24 h before EcoHIV infection, euthanized 3 days later, and spleens collected for measurement of C. total, D. integrated, and E. 2-LTR circle vDNA; F. spliced vRNA by QPCR of DNA or cDNA as described in Methods.

    Article Snippet: DNA from human cells was standardized by QPCR amplification of human β-globin QPCR (ThermoFisher Scientific) and custom-designed mouse β-globin QPCR [ ].

    Techniques: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Generated, Amplification, Mouse Assay, Infection

    Both EcoHIV and MLV infect mouse brain but only EcoHIV-infected mice develop cognitive impairment. A. Design of the experiment. B. Groups of mice were gavaged with cART or vehicle before and during infection with indicated viruses or vehicle. At day 21 after infection, mice were tested for errors (left panel) and latency (middle panel) in finding the hidden platform, the right panel shows latency to reach the visible platform. RT represents the retention trial. C. Two days after completion of RAWM, the same mice were used to test contextual response to fear conditioning training. The percentage of freezing time was calculated for contextual fear memory deficit in each group of mice. D. Mice were euthanized on day 40 after infection and levels of total vDNA in spleen and brain measured by QPCR (left panel) and integrated viral DNA measured by nested QPCR in PM (right panel). All data are mean ± standard errors. * P

    Journal: PLoS Pathogens

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

    doi: 10.1371/journal.ppat.1007061

    Figure Lengend Snippet: Both EcoHIV and MLV infect mouse brain but only EcoHIV-infected mice develop cognitive impairment. A. Design of the experiment. B. Groups of mice were gavaged with cART or vehicle before and during infection with indicated viruses or vehicle. At day 21 after infection, mice were tested for errors (left panel) and latency (middle panel) in finding the hidden platform, the right panel shows latency to reach the visible platform. RT represents the retention trial. C. Two days after completion of RAWM, the same mice were used to test contextual response to fear conditioning training. The percentage of freezing time was calculated for contextual fear memory deficit in each group of mice. D. Mice were euthanized on day 40 after infection and levels of total vDNA in spleen and brain measured by QPCR (left panel) and integrated viral DNA measured by nested QPCR in PM (right panel). All data are mean ± standard errors. * P

    Article Snippet: DNA from human cells was standardized by QPCR amplification of human β-globin QPCR (ThermoFisher Scientific) and custom-designed mouse β-globin QPCR [ ].

    Techniques: Infection, Mouse Assay, Real-time Polymerase Chain Reaction

    EcoHIV establishes latent reservoirs in resting CD4 + cells that are inducible by epigenetic modulators. A. CD4 + T cells isolated from spleen at day 25 post-infection were cocultured with uninfected cells in the presence or absence of ABC as indicated prior to vDNA amplification, each symbol represents culture from a single mouse. B. Six weeks after EcoHIV infection, CD4 + splenic T cells were harvested and purified from donor male 129X1/SvJ mice and injected into the recipient female 129X1 nude mice; their PM were collected after one week. Y chromosome and EcoHIV gag RNA levels in PM were determined by QPCR. Each symbol represents a single mouse. C.-D. Twelve weeks after infection, resting CD4 + T cells were purified from the spleen and C. The levels of integrated EcoHIV DNA and D. Genomic RNA were measured by QPCR. E. Eight weeks after EcoHIV infection of mice, resting CD4 + T cells were isolated and exposed to the agents indicated in culture for 2 days prior to collection and measurement of EcoHIV mRNA QPCR. F. Six weeks after infection by EcoHIV-Luc, mice were treated as shown, euthanized and splenic CD4 + cells isolated for measurement of virus RNA expression (left panel) or protein expression (right panel). * P

    Journal: PLoS Pathogens

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

    doi: 10.1371/journal.ppat.1007061

    Figure Lengend Snippet: EcoHIV establishes latent reservoirs in resting CD4 + cells that are inducible by epigenetic modulators. A. CD4 + T cells isolated from spleen at day 25 post-infection were cocultured with uninfected cells in the presence or absence of ABC as indicated prior to vDNA amplification, each symbol represents culture from a single mouse. B. Six weeks after EcoHIV infection, CD4 + splenic T cells were harvested and purified from donor male 129X1/SvJ mice and injected into the recipient female 129X1 nude mice; their PM were collected after one week. Y chromosome and EcoHIV gag RNA levels in PM were determined by QPCR. Each symbol represents a single mouse. C.-D. Twelve weeks after infection, resting CD4 + T cells were purified from the spleen and C. The levels of integrated EcoHIV DNA and D. Genomic RNA were measured by QPCR. E. Eight weeks after EcoHIV infection of mice, resting CD4 + T cells were isolated and exposed to the agents indicated in culture for 2 days prior to collection and measurement of EcoHIV mRNA QPCR. F. Six weeks after infection by EcoHIV-Luc, mice were treated as shown, euthanized and splenic CD4 + cells isolated for measurement of virus RNA expression (left panel) or protein expression (right panel). * P

    Article Snippet: DNA from human cells was standardized by QPCR amplification of human β-globin QPCR (ThermoFisher Scientific) and custom-designed mouse β-globin QPCR [ ].

    Techniques: Isolation, Infection, Amplification, Purification, Mouse Assay, Injection, Real-time Polymerase Chain Reaction, RNA Expression, Expressing

    Mouse macrophages are susceptible to EcoHIV but not MLV. A. Expression of CAT-1 was determined in peritoneal cells (PC) and thymus (TH) of C57BL/6 mice by QPCR. B. Ten days after EcoHIV or MLV infection of mice, the indicated tissues were harvested for measurement of viral nucleic acids by QPCR. C. Eight weeks after mouse infection by EcoHIV or MLV, F4/80 + macrophages were purified from PC and integrated viral DNA in unfractionated or F4/80 + PC was measured by nested-QPCR. D. C57BL/6 euthymic and athymic mice were infected by EcoHIV and PC harvested from groups at the times indicated to measure spliced EcoHIV RNA. Mean and standard errors are shown (* P

    Journal: PLoS Pathogens

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

    doi: 10.1371/journal.ppat.1007061

    Figure Lengend Snippet: Mouse macrophages are susceptible to EcoHIV but not MLV. A. Expression of CAT-1 was determined in peritoneal cells (PC) and thymus (TH) of C57BL/6 mice by QPCR. B. Ten days after EcoHIV or MLV infection of mice, the indicated tissues were harvested for measurement of viral nucleic acids by QPCR. C. Eight weeks after mouse infection by EcoHIV or MLV, F4/80 + macrophages were purified from PC and integrated viral DNA in unfractionated or F4/80 + PC was measured by nested-QPCR. D. C57BL/6 euthymic and athymic mice were infected by EcoHIV and PC harvested from groups at the times indicated to measure spliced EcoHIV RNA. Mean and standard errors are shown (* P

    Article Snippet: DNA from human cells was standardized by QPCR amplification of human β-globin QPCR (ThermoFisher Scientific) and custom-designed mouse β-globin QPCR [ ].

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

    EcoHIV infects mice and induces antiviral immune responses but not immunodeficiency in mice. A.-B. Kinetics of production of total EcoHIV DNA (left panels), integrated EcoHIV DNA (middle panels), and genomic EcoHIV RNA (right panels) were measured by QPCR in A. spleen and B. PC at the times indicated after infection. Each point represents a single mouse. C. EcoHIV gag RNA burden at each time point in copies per ml of whole blood was measured at the times after infection indicated. The dashed line indicates limit of detection of 10 copies. D. The frequency of interferon-γ expression by CD4 + or CD8 + spleen cells at the time indicated after EcoHIV or mock infection was measured by flow cytometry. E. The number of CD4 + and CD8 + T cells was measured by flow cytometry at the indicated times and the CD4 + :CD8 + ratio is shown. Symbols represent individual mice, the horizontal lines represent the mean. F. Longitudinal anti-NDK Gag antibodies in 3 EcoHIV-infected mice were measured by ELISA at the times indicated after infection. Each panel represents titers in a single mouse, mean +/- standard errors are shown.

    Journal: PLoS Pathogens

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

    doi: 10.1371/journal.ppat.1007061

    Figure Lengend Snippet: EcoHIV infects mice and induces antiviral immune responses but not immunodeficiency in mice. A.-B. Kinetics of production of total EcoHIV DNA (left panels), integrated EcoHIV DNA (middle panels), and genomic EcoHIV RNA (right panels) were measured by QPCR in A. spleen and B. PC at the times indicated after infection. Each point represents a single mouse. C. EcoHIV gag RNA burden at each time point in copies per ml of whole blood was measured at the times after infection indicated. The dashed line indicates limit of detection of 10 copies. D. The frequency of interferon-γ expression by CD4 + or CD8 + spleen cells at the time indicated after EcoHIV or mock infection was measured by flow cytometry. E. The number of CD4 + and CD8 + T cells was measured by flow cytometry at the indicated times and the CD4 + :CD8 + ratio is shown. Symbols represent individual mice, the horizontal lines represent the mean. F. Longitudinal anti-NDK Gag antibodies in 3 EcoHIV-infected mice were measured by ELISA at the times indicated after infection. Each panel represents titers in a single mouse, mean +/- standard errors are shown.

    Article Snippet: DNA from human cells was standardized by QPCR amplification of human β-globin QPCR (ThermoFisher Scientific) and custom-designed mouse β-globin QPCR [ ].

    Techniques: Mouse Assay, Real-time Polymerase Chain Reaction, Infection, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    EcoHIV integrates efficiently in the murine host genome. A. Schematic representation of strategy for assay of integration. In the first round PCR, primers, B1-F and B1-R, from the host B1 repetitive elements and a HIV-specific primer, Fnef, were used to amplify a region of integrated virus and the host flanking region. The second nested QPCR based on the HIV RU5 region was performed using the preamplified products. B. Integration standard curve was generated by logarithmic regression of the amplified IS sample signals. C.-F. Mice (n = 4/system) were pretreated with ABC or RAL 24 h before EcoHIV infection, euthanized 3 days later, and spleens collected for measurement of C. total, D. integrated, and E. 2-LTR circle vDNA; F. spliced vRNA by QPCR of DNA or cDNA as described in Methods.

    Journal: PLoS Pathogens

    Article Title: EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

    doi: 10.1371/journal.ppat.1007061

    Figure Lengend Snippet: EcoHIV integrates efficiently in the murine host genome. A. Schematic representation of strategy for assay of integration. In the first round PCR, primers, B1-F and B1-R, from the host B1 repetitive elements and a HIV-specific primer, Fnef, were used to amplify a region of integrated virus and the host flanking region. The second nested QPCR based on the HIV RU5 region was performed using the preamplified products. B. Integration standard curve was generated by logarithmic regression of the amplified IS sample signals. C.-F. Mice (n = 4/system) were pretreated with ABC or RAL 24 h before EcoHIV infection, euthanized 3 days later, and spleens collected for measurement of C. total, D. integrated, and E. 2-LTR circle vDNA; F. spliced vRNA by QPCR of DNA or cDNA as described in Methods.

    Article Snippet: DNA from human cells was standardized by QPCR amplification of human β-globin QPCR (ThermoFisher Scientific) and custom-designed mouse β-globin QPCR [ ].

    Techniques: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Generated, Amplification, Mouse Assay, Infection

    (A) mRNA level of TACC3 was examined in osteosarcoma cell lines using RT-qPCR. (B) Protein levels of TACC3 were determined by western blot analysis in various osteosarcoma cell lines. (C) The mRNA level of TACC3 in osteosarcoma tissues and normal matched non-cancerous tissue as assessed by RT-qPCR. NS, non-cancerous tissue; OS, osteosarcoma; TACC3, transforming acidic coiled-coil protein; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

    Journal: Oncology Letters

    Article Title: Downregulation of TACC3 inhibits tumor growth and migration in osteosarcoma cells through regulation of the NF-κB signaling pathway

    doi: 10.3892/ol.2018.8262

    Figure Lengend Snippet: (A) mRNA level of TACC3 was examined in osteosarcoma cell lines using RT-qPCR. (B) Protein levels of TACC3 were determined by western blot analysis in various osteosarcoma cell lines. (C) The mRNA level of TACC3 in osteosarcoma tissues and normal matched non-cancerous tissue as assessed by RT-qPCR. NS, non-cancerous tissue; OS, osteosarcoma; TACC3, transforming acidic coiled-coil protein; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

    Article Snippet: The primers used were as follows: TACC3 forward, 5′-CCTCTTCAAGCGTTTTGAGAAAC-3′ and reverse, 5′-GCCCTCCTGGGTGATCCTT-3′; GAPDH, forward, 5′-CTCCTCCTGTTCGACAGTCAGC-3′, and reverse, 5′-CCCAATACGACCAAATCCGTT-3′. qPCR amplification was performed in an ABI 7900HT Real-time PCR system (Thermo Fisher Scientific, Inc.).

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