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  • 99
    Thermo Fisher superscript first strand synthesis system
    Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using <t>Superscript™</t> <t>First-Strand</t> <t>Synthesis</t> <t>System</t> with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P
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    99
    Millipore rt pcr
    Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using <t>Superscript™</t> <t>First-Strand</t> <t>Synthesis</t> <t>System</t> with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P
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    99
    Thermo Fisher rt pcr
    Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using <t>Superscript™</t> <t>First-Strand</t> <t>Synthesis</t> <t>System</t> with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P
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    Qiagen onestep rt pcr kit
    Validation of model predictions using targeted amplification of editable sites from single cells. ( a ) Wiggle plots showing coverage in 3′-untranslated regions for B2m, Anxa5 and Cybb in the 24 bone marrow-derived macrophages profiled. ( b – d ) Sequence alignments from targeted <t>RT–PCR</t> amplification and Sanger sequencing of bacterial colonies for ( b ) B2m, ( c ) Anxa5 and ( d ) Cybb transcripts from gDNA and cDNA from a bulk sample (amplified using standard PCR), and cDNA of single cells (amplified using a modified <t>OneStep</t> RT–PCR protocol, per Supplementary Fig. 4 ). Alignments, showing the sequence space surrounding a particular editable site, are clustered by sample. Alignments are colour-coded to indicate whether the sequence aligned contained (red) or lacked (grey) editing in the length of the amplicon. Though a C-to-U change may not be shown in the narrow window illustrated, a red sequence would indicate that the amplicon sequence contained at least one C-to-U edit elsewhere (red). Lack of editing in the gDNA indicates that the C-to-U transitions observed are bona fide APOBEC1-mediated RNA editing events.
    Onestep Rt Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 14578 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Bio-Rad rt pcr
    Validation of model predictions using targeted amplification of editable sites from single cells. ( a ) Wiggle plots showing coverage in 3′-untranslated regions for B2m, Anxa5 and Cybb in the 24 bone marrow-derived macrophages profiled. ( b – d ) Sequence alignments from targeted <t>RT–PCR</t> amplification and Sanger sequencing of bacterial colonies for ( b ) B2m, ( c ) Anxa5 and ( d ) Cybb transcripts from gDNA and cDNA from a bulk sample (amplified using standard PCR), and cDNA of single cells (amplified using a modified <t>OneStep</t> RT–PCR protocol, per Supplementary Fig. 4 ). Alignments, showing the sequence space surrounding a particular editable site, are clustered by sample. Alignments are colour-coded to indicate whether the sequence aligned contained (red) or lacked (grey) editing in the length of the amplicon. Though a C-to-U change may not be shown in the narrow window illustrated, a red sequence would indicate that the amplicon sequence contained at least one C-to-U edit elsewhere (red). Lack of editing in the gDNA indicates that the C-to-U transitions observed are bona fide APOBEC1-mediated RNA editing events.
    Rt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 95/100, based on 3207 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher superscript iii one step rt pcr system
    PRL-2 mRNA and protein levels were significantly suppressed in the PRL-2 knockdown cells The cell clone numbers 376 and 441 indicate the starting nucleotide number of siRNA- or shRNA-targeting sequences of PRL-2 mRNA. <t>RT-PCR</t> and Western blotting images were processed and quantified with Fuji Multi Gauge software (Fujifilm). A) RT-PCR demonstrated that PRL-2 was over-expressed in several lung cancer cells including A549 cells. β-actin was used as internal control. B) PRL-2 was selectively down-regulated at the mRNA level in cells with transient and stable PRL-2 knockdown. The siRNA transiently transfected cells displayed ~80% decrease in PRL-2 mRNA level, while there was a ~50% decrease in the shRNA transfected stable knockdown cells. β-actin was used as the internal control to determine expression levels. The average fold changes, as shown above the blot, were calculated from <t>three</t> independent experiments and normalized to relative PRL-2 levels in the parental A549 cells. C) PRL-2 protein level was selectively down-regulated. The upper blot was obtained with an antibody from R D Systems (MAB32191) that recognizes all three PRL proteins and the middle blot was produced with a PRL-2 specific polyclonal antibody from Bethyl (BL1205). β tubulin was used as an internal control.
    Superscript Iii One Step Rt Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8429 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad real time reverse transcription polymerase chain reaction
    Effect of nicotine on mRNA abundance of AT 1 R and AT 2 R in aortic rings in adult offspring. RNA was extracted from aortic rings isolated from saline control and nicotine-treated animals. The abundance of AT 1a R, AT 1b R, and AT 2 R mRNA was determined by <t>real-time</t> <t>reverse</t> <t>transcription-polymerase</t> <t>chain</t> <t>reaction</t> analysis (as described in METHODS ). Data are means ± SEM of tissues from saline control (n = 4) and nicotine (n = 4). The sample sizes represent the animal number from different litters of each group. Data were analyzed by student t -test. * P
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    Thermo Fisher thermoscript rt pcr system
    Effect of nicotine on mRNA abundance of AT 1 R and AT 2 R in aortic rings in adult offspring. RNA was extracted from aortic rings isolated from saline control and nicotine-treated animals. The abundance of AT 1a R, AT 1b R, and AT 2 R mRNA was determined by <t>real-time</t> <t>reverse</t> <t>transcription-polymerase</t> <t>chain</t> <t>reaction</t> analysis (as described in METHODS ). Data are means ± SEM of tissues from saline control (n = 4) and nicotine (n = 4). The sample sizes represent the animal number from different litters of each group. Data were analyzed by student t -test. * P
    Thermoscript Rt Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 8191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen quantitect sybr green rt pcr kit
    T m analysis of real-time <t>PCR</t> with <t>SYBR</t> Green as fluorescent dye. T m analysis allows the differentiation of specific PCR product (amplicon) from non-specific amplification products, such as primer-dimers. Samples: 1 to 7, patient samples (stool); 8, negative control (water); 9, positive control (NoV positive stool). Amplified products from NoV template RNA can be reliably distinguished from non-specific products by different melting points. The melting temperature for the positive samples was 79.0 +/- 0.5°C. Non-specific products melt at lower temperatures.
    Quantitect Sybr Green Rt Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 8359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bio-Rad quantitative reverse transcription polymerase chain reaction
    SB suppresses TPA-induced inflammation. ( A ) Effect of SB on chronic TPA-induced hyperplasia (top) proliferation (middle) and apoptosis (bottom) ( B ) Decreased cutaneous neutrophils (MPO+ cells) after acute and chronic TPA + SB treatment. ( C ) Decreased keratinocyte chemokine (KC) and macrophage-inflammatory protein 2 (MIP2) gene expression in acute and chronically SB- and TPA-treated whole skin. Results normalized to TPA only samples. <t>Reverse</t> <t>transcription–polymerase</t> <t>chain</t> <t>reaction</t> showing induction of these chemokines by TPA (bottom). n = 4–6 mice per treatment group. A, acetone.
    Quantitative Reverse Transcription Polymerase Chain Reaction, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rt pcr total rna
    Ig gene rearrangement and transcription in μMT mice liver epithelial cells. ( A ) Northern blot analysis. Total <t>RNA</t> from liver cells of three μMT mice and WT spleen cells (positive control) were transferred to nylon membrane, and probed with probes to constant region of Igμ and Igκ; 28 s and 18 s RNA were shown by DNA agarose gel electrophoresis. ( B ) In situ hybridization analysis was shown for detection of Ig μ and Ig κ transcripts in μMT mice with antisense or sense probes for C region of μ chain or J-C region of κ chain. ( C ) a It was shown for the primer design of Ig μ variable region, Ig κ variable region and Igλ; b Sorted liver epithelial cells from two μMT mice were analyzed for Ig rearrangement and transcription by <t>RT-PCR.</t> WT spleen cells were used as the positive control. Variable region and constant region of heavy chain (μ, α, δ, γ, ε) and light chain (κ, λ), CD20 and GAPDH were amplified; “1, 2” represented two samples of sorted μMT liver epithelial cells.
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    TaKaRa primescript rt pcr kit
    Ig gene rearrangement and transcription in μMT mice liver epithelial cells. ( A ) Northern blot analysis. Total <t>RNA</t> from liver cells of three μMT mice and WT spleen cells (positive control) were transferred to nylon membrane, and probed with probes to constant region of Igμ and Igκ; 28 s and 18 s RNA were shown by DNA agarose gel electrophoresis. ( B ) In situ hybridization analysis was shown for detection of Ig μ and Ig κ transcripts in μMT mice with antisense or sense probes for C region of μ chain or J-C region of κ chain. ( C ) a It was shown for the primer design of Ig μ variable region, Ig κ variable region and Igλ; b Sorted liver epithelial cells from two μMT mice were analyzed for Ig rearrangement and transcription by <t>RT-PCR.</t> WT spleen cells were used as the positive control. Variable region and constant region of heavy chain (μ, α, δ, γ, ε) and light chain (κ, λ), CD20 and GAPDH were amplified; “1, 2” represented two samples of sorted μMT liver epithelial cells.
    Primescript Rt Pcr Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 4795 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad iscript one step rt pcr kit
    In utero B(a)P-exposure upregulates heart nNOS, eNOS, BH4/BH2 oxidore-ductase and AngII mRNA in offspring. Relative expression levels of nNOS, eNOS, BH4/BH2 and AngII as measured by <t>qRT-PCR.</t> Quantitative RT-PCR was performed using <t>iScript</t> one-step RT-PCR
    Iscript One Step Rt Pcr Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 5922 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega access rt pcr system
    Nectin-4 and Nectin-1 are expressed in human mesothelial cells and ovarian cancer patient samples <t>RT-PCR</t> analysis of Nectin-4 and Nectin-1 expression in human mesothelial cell lines LP9 and Met5a, and matched samples from four patients with high grade serous ovarian cancer: ascites cells (As), primary ovarian tumor (Ov), and omental metastases (Om). Nectin-4 <t>RNA</t> was expressed in all of the samples, at variable levels. Nectin-1 RNA was more consistently expressed across samples. β-actin, loading control.
    Access Rt Pcr System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3466 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen quantitect probe rt pcr kit
    Establishment and validation of multivirus real‐time <t>PCR.</t> A : Procedure of the multivirus real‐time PCR system. DNA sample was mixed with <t>Quantitect</t> 2× master mix, and RNA sample was mixed with Quantitect 2× master mix and reverse transcriptase (RT) mix. These mixtures were poured into each well in a 96‐well plate at 10 µl per well. Ten microliters of 2× probe and primer mix were then added to each well in a premixed 96‐well plate. Finally, the virus genes were amplified and detected in a real‐time PCR machine for 2 hr. B : Validation of the multivirus real‐time PCR. A gene expression image by TreeView software based on the results of the multivirus real‐time PCR for control samples is shown. A horizontal line shows each probe–primer set and a vertical line is one sample of positive control. Gray vertical lines indicate no sample. A scale bar indicates copy number of color. A green box indicates specific reactions of target positive controls in specific probe–primer sets. Arrows of (a–c) also show specific signals. The arrow (a) shows positive signal for TTV in a brain sample with both JCV and TTV infection. The arrows (b1–3) show that a probe–primer set for pan‐enterovirus reacted with poliovirus (b1), Coxsackievirus B3 (b2), and Echovirus 6 (b3) positive samples. The arrow (c) shows that a probe–primer set for influenza virus A reacted with H5N1 influenza virus. Details of positive controls were listed in Supplementary Table II.
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    Thermo Fisher superscript one step rt pcr
    FatB was expressed in B. thuringiensis cells grown in iron restricted medium at 25 °C, 30 °C and 37 °C. <t>RNA</t> was isolated from stationary phase cells and reverse transcription <t>PCR</t> with 35 cycles was used to detect gene expression. Experiments were performed in triplicate and a representative gel is shown.
    Superscript One Step Rt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3306 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa rt pcr kit
    Expression analysis of eight Arabidopsis genes. A, Total <t>RNA</t> was isolated from root (R), hypocotyl (H), cotyledon (C), leaf (L), and stem (S) tissues of 3-week-old in vitro-germinated plants and cell suspension cultures (Cs). <t>RT-PCR</t> was performed on equal
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    Stratagene rt pcr
    Expression analysis of eight Arabidopsis genes. A, Total <t>RNA</t> was isolated from root (R), hypocotyl (H), cotyledon (C), leaf (L), and stem (S) tissues of 3-week-old in vitro-germinated plants and cell suspension cultures (Cs). <t>RT-PCR</t> was performed on equal
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    Qiagen quantifast sybr green rt pcr kit
    Compensatory analysis of miR21 knock-down targeted to miR21 primary transcript by snoMEN vector. ( a ) Micrographs showing rescue experiment of apoptosis induction by co-transfecting with pri-miR21-snoMEN (mCherry) and pre-miR21 expression plasmid (GFP+miR21)/Control plasmid that expresses GFP protein alone (GFP). Images were taken 72 hours followed after transfection. Scale bar is 10 μm. ( b ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after co-transfection with pri-miR21-snoMEN (mCherry) and pre-miR21 expression plasmid (miR21)/Control plasmid that express GFP protein alone (GFP) and <t>qRT-PCR</t> was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21, Red). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta <t>SYBR</t> Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21, Green). U3 snoRNA was used as a control. Graph depicts mean and standard deviation from a minimum of 3 independent experiments. ( c ) Micrographs show prevention of non-apoptosis induction by transfecting with pri-miR21-snoMEN mut, which has 3 base mismatches in each M box complementary sequence to pri-miR21. Images were taken 72 hours followed after transfection. Scale bar is 10 μm. ( d ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after transfection with either pri-miR21-snoMEN mut or Control. Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ). U3 snoRNA was used as a control. Graph depicts mean and standard deviation from a minimum of 4 independent experiments.
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    Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P

    Journal: BMC Medical Genomics

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

    doi: 10.1186/1755-8794-1-6

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

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

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

    Transcript levels of AGO and other small RNA biogenesis genes. Three different life stages (myc, mycelium; zoo, zoospores; gc, germinated cysts) and an infection time series (3, 6, 12, 24, and 48 h post infection) were assayed in replicate by qRT-PCR to determine transcript levels of (A) DCL 1, DCL 2, RDR , and (B,C) the AGO homologs in P. sojae as compared to housekeeping genes b-tubulin and WS41 . Each life stage or time series point is normalized to the housekeeping gene(s). Standard error bars are included, but in most cases the standard error bars are blocked by the symbol due to low value.

    Journal: Frontiers in Microbiology

    Article Title: Expansion and Divergence of Argonaute Genes in the Oomycete Genus Phytophthora

    doi: 10.3389/fmicb.2018.02841

    Figure Lengend Snippet: Transcript levels of AGO and other small RNA biogenesis genes. Three different life stages (myc, mycelium; zoo, zoospores; gc, germinated cysts) and an infection time series (3, 6, 12, 24, and 48 h post infection) were assayed in replicate by qRT-PCR to determine transcript levels of (A) DCL 1, DCL 2, RDR , and (B,C) the AGO homologs in P. sojae as compared to housekeeping genes b-tubulin and WS41 . Each life stage or time series point is normalized to the housekeeping gene(s). Standard error bars are included, but in most cases the standard error bars are blocked by the symbol due to low value.

    Article Snippet: The Superscript III First-Strand Synthesis System for RT-PCR kit (Invitrogen, Carlsbad, CA, United States) was used to produce cDNA, followed with purification by phenol:chloroform extraction. cDNA was quantified with a Nanodrop ND-1000 (Thermo Scientific, Waltham, MA, United States) to allow for equal quantities of cDNA template in subsequent reactions.

    Techniques: Infection, Quantitative RT-PCR

    Analysis of the 5′ end of the glpFK message. (A) Gel electrophoresis of PCR products of the 5′ end of the glpFK cDNA. RNA ligase-mediated RT–PCR was employed to amplify the 5′ end of the glpFK mRNA. cDNA was synthesized using an Invitrogen superscript first-strand synthesis kit. The arrow points to the product resulting from a newly initiated message. The other band is a nonspecific PCR product. (B) Chromatogram of a part of the DNA sequence showing the junction between the 5′ end of the glpFK cDNA and the reverse transcribed adaptor. The amplified 5′ end of the glpFK cDNA was sequenced using the oligo PglpFK-extn-R (see Table S2 ) that binds to the ∼210 bp region downstream of +1. The arrow points to the transcriptional start site (+1) on the complementary strand.

    Journal: PLoS Genetics

    Article Title: A Novel Mechanism of Transposon-Mediated Gene Activation

    doi: 10.1371/journal.pgen.1000689

    Figure Lengend Snippet: Analysis of the 5′ end of the glpFK message. (A) Gel electrophoresis of PCR products of the 5′ end of the glpFK cDNA. RNA ligase-mediated RT–PCR was employed to amplify the 5′ end of the glpFK mRNA. cDNA was synthesized using an Invitrogen superscript first-strand synthesis kit. The arrow points to the product resulting from a newly initiated message. The other band is a nonspecific PCR product. (B) Chromatogram of a part of the DNA sequence showing the junction between the 5′ end of the glpFK cDNA and the reverse transcribed adaptor. The amplified 5′ end of the glpFK cDNA was sequenced using the oligo PglpFK-extn-R (see Table S2 ) that binds to the ∼210 bp region downstream of +1. The arrow points to the transcriptional start site (+1) on the complementary strand.

    Article Snippet: The contaminated chromosomal DNA in RNA samples was removed by DNase I treatment. cDNAs were synthesized using an Invitrogen superscript first-strand synthesis kit. polA , encoding DNA polymerase I, was included as an internal control .

    Techniques: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Synthesized, Sequencing, Amplification

    Validation of model predictions using targeted amplification of editable sites from single cells. ( a ) Wiggle plots showing coverage in 3′-untranslated regions for B2m, Anxa5 and Cybb in the 24 bone marrow-derived macrophages profiled. ( b – d ) Sequence alignments from targeted RT–PCR amplification and Sanger sequencing of bacterial colonies for ( b ) B2m, ( c ) Anxa5 and ( d ) Cybb transcripts from gDNA and cDNA from a bulk sample (amplified using standard PCR), and cDNA of single cells (amplified using a modified OneStep RT–PCR protocol, per Supplementary Fig. 4 ). Alignments, showing the sequence space surrounding a particular editable site, are clustered by sample. Alignments are colour-coded to indicate whether the sequence aligned contained (red) or lacked (grey) editing in the length of the amplicon. Though a C-to-U change may not be shown in the narrow window illustrated, a red sequence would indicate that the amplicon sequence contained at least one C-to-U edit elsewhere (red). Lack of editing in the gDNA indicates that the C-to-U transitions observed are bona fide APOBEC1-mediated RNA editing events.

    Journal: Nature Communications

    Article Title: RNA editing generates cellular subsets with diverse sequence within populations

    doi: 10.1038/ncomms12145

    Figure Lengend Snippet: Validation of model predictions using targeted amplification of editable sites from single cells. ( a ) Wiggle plots showing coverage in 3′-untranslated regions for B2m, Anxa5 and Cybb in the 24 bone marrow-derived macrophages profiled. ( b – d ) Sequence alignments from targeted RT–PCR amplification and Sanger sequencing of bacterial colonies for ( b ) B2m, ( c ) Anxa5 and ( d ) Cybb transcripts from gDNA and cDNA from a bulk sample (amplified using standard PCR), and cDNA of single cells (amplified using a modified OneStep RT–PCR protocol, per Supplementary Fig. 4 ). Alignments, showing the sequence space surrounding a particular editable site, are clustered by sample. Alignments are colour-coded to indicate whether the sequence aligned contained (red) or lacked (grey) editing in the length of the amplicon. Though a C-to-U change may not be shown in the narrow window illustrated, a red sequence would indicate that the amplicon sequence contained at least one C-to-U edit elsewhere (red). Lack of editing in the gDNA indicates that the C-to-U transitions observed are bona fide APOBEC1-mediated RNA editing events.

    Article Snippet: RT–PCR amplification was done with gene specific primers and the OneStep RT–PCR kit (Qiagen), using a modified protocol.

    Techniques: Amplification, Derivative Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Modification

    Nested PCR for WT1 expression.

    Journal: Biomarker Insights

    Article Title: Studies of Wilms' Tumor (WT1) Gene Expression in Adult Acute Leukemias in Singapore

    doi:

    Figure Lengend Snippet: Nested PCR for WT1 expression.

    Article Snippet: Nested RT-PCR (reverse-transcription PCR) One step nested reverse transcription polymerase chain reaction (Nested RT-PCR) was performed by using a one-step RT-PCR kit. (Qiagen, Valencia, CA, USA) WT1 exon 1–4 was amplified using forward (5′-CCTACCTGCCC AG CTGCCTC-3′) and reverse (5′-CTCCTAAGTTCATCTGATTCC-3′) primers for 20 cycles (annealing temperature 56 °C), followed by nested PCR (forward: 5′-AGAGCCAGCCCGCTATTCG-3′; and reverse: GGTCATGCATTCAAGCTGG-3′ primers) for 30 cycles (annealing temperature 58 °C).

    Techniques: Nested PCR, Expressing

    PRL-2 mRNA and protein levels were significantly suppressed in the PRL-2 knockdown cells The cell clone numbers 376 and 441 indicate the starting nucleotide number of siRNA- or shRNA-targeting sequences of PRL-2 mRNA. RT-PCR and Western blotting images were processed and quantified with Fuji Multi Gauge software (Fujifilm). A) RT-PCR demonstrated that PRL-2 was over-expressed in several lung cancer cells including A549 cells. β-actin was used as internal control. B) PRL-2 was selectively down-regulated at the mRNA level in cells with transient and stable PRL-2 knockdown. The siRNA transiently transfected cells displayed ~80% decrease in PRL-2 mRNA level, while there was a ~50% decrease in the shRNA transfected stable knockdown cells. β-actin was used as the internal control to determine expression levels. The average fold changes, as shown above the blot, were calculated from three independent experiments and normalized to relative PRL-2 levels in the parental A549 cells. C) PRL-2 protein level was selectively down-regulated. The upper blot was obtained with an antibody from R D Systems (MAB32191) that recognizes all three PRL proteins and the middle blot was produced with a PRL-2 specific polyclonal antibody from Bethyl (BL1205). β tubulin was used as an internal control.

    Journal: Oncogene

    Article Title: Metastasis-associated phosphatase PRL-2 regulates tumor cell migration and invasion

    doi: 10.1038/onc.2011.281

    Figure Lengend Snippet: PRL-2 mRNA and protein levels were significantly suppressed in the PRL-2 knockdown cells The cell clone numbers 376 and 441 indicate the starting nucleotide number of siRNA- or shRNA-targeting sequences of PRL-2 mRNA. RT-PCR and Western blotting images were processed and quantified with Fuji Multi Gauge software (Fujifilm). A) RT-PCR demonstrated that PRL-2 was over-expressed in several lung cancer cells including A549 cells. β-actin was used as internal control. B) PRL-2 was selectively down-regulated at the mRNA level in cells with transient and stable PRL-2 knockdown. The siRNA transiently transfected cells displayed ~80% decrease in PRL-2 mRNA level, while there was a ~50% decrease in the shRNA transfected stable knockdown cells. β-actin was used as the internal control to determine expression levels. The average fold changes, as shown above the blot, were calculated from three independent experiments and normalized to relative PRL-2 levels in the parental A549 cells. C) PRL-2 protein level was selectively down-regulated. The upper blot was obtained with an antibody from R D Systems (MAB32191) that recognizes all three PRL proteins and the middle blot was produced with a PRL-2 specific polyclonal antibody from Bethyl (BL1205). β tubulin was used as an internal control.

    Article Snippet: Reverse transcription-PCR (RT-PCR) for PRL-1, PRL-2, PRL-3, and actin as an internal control was carried out in a volume of 50 µL by SuperScript III One-step RT-PCR System (Invitrogen) as per manufacturer's instruction.

    Techniques: shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Software, Transfection, Expressing, Produced

    Effect of nicotine on mRNA abundance of AT 1 R and AT 2 R in aortic rings in adult offspring. RNA was extracted from aortic rings isolated from saline control and nicotine-treated animals. The abundance of AT 1a R, AT 1b R, and AT 2 R mRNA was determined by real-time reverse transcription-polymerase chain reaction analysis (as described in METHODS ). Data are means ± SEM of tissues from saline control (n = 4) and nicotine (n = 4). The sample sizes represent the animal number from different litters of each group. Data were analyzed by student t -test. * P

    Journal: PLoS ONE

    Article Title: Perinatal Nicotine Exposure Increases Angiotensin II Receptor-Mediated Vascular Contractility in Adult Offspring

    doi: 10.1371/journal.pone.0108161

    Figure Lengend Snippet: Effect of nicotine on mRNA abundance of AT 1 R and AT 2 R in aortic rings in adult offspring. RNA was extracted from aortic rings isolated from saline control and nicotine-treated animals. The abundance of AT 1a R, AT 1b R, and AT 2 R mRNA was determined by real-time reverse transcription-polymerase chain reaction analysis (as described in METHODS ). Data are means ± SEM of tissues from saline control (n = 4) and nicotine (n = 4). The sample sizes represent the animal number from different litters of each group. Data were analyzed by student t -test. * P

    Article Snippet: Real-Time Reverse Transcription–Polymerase Chain Reaction (RT-PCR) RNA was extracted from aortic rings and abundance of AT1a R, AT1b R, and AT2 R mRNA was determined by real-time reverse transcription–polymerase chain reaction using an Icycler Thermal cycler (Bio-Rad, Hercules, CA), as described previously , .

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction

    T m analysis of real-time PCR with SYBR Green as fluorescent dye. T m analysis allows the differentiation of specific PCR product (amplicon) from non-specific amplification products, such as primer-dimers. Samples: 1 to 7, patient samples (stool); 8, negative control (water); 9, positive control (NoV positive stool). Amplified products from NoV template RNA can be reliably distinguished from non-specific products by different melting points. The melting temperature for the positive samples was 79.0 +/- 0.5°C. Non-specific products melt at lower temperatures.

    Journal: BMC Infectious Diseases

    Article Title: Fast detection of Noroviruses using a real-time PCR assay and automated sample preparation

    doi: 10.1186/1471-2334-4-15

    Figure Lengend Snippet: T m analysis of real-time PCR with SYBR Green as fluorescent dye. T m analysis allows the differentiation of specific PCR product (amplicon) from non-specific amplification products, such as primer-dimers. Samples: 1 to 7, patient samples (stool); 8, negative control (water); 9, positive control (NoV positive stool). Amplified products from NoV template RNA can be reliably distinguished from non-specific products by different melting points. The melting temperature for the positive samples was 79.0 +/- 0.5°C. Non-specific products melt at lower temperatures.

    Article Snippet: QuantiTect™ SYBR® Green RT-PCR kit was purchased from Qiagen, (Qiagen, Hilden, Germany).

    Techniques: Real-time Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction, Amplification, Negative Control, Positive Control

    SB suppresses TPA-induced inflammation. ( A ) Effect of SB on chronic TPA-induced hyperplasia (top) proliferation (middle) and apoptosis (bottom) ( B ) Decreased cutaneous neutrophils (MPO+ cells) after acute and chronic TPA + SB treatment. ( C ) Decreased keratinocyte chemokine (KC) and macrophage-inflammatory protein 2 (MIP2) gene expression in acute and chronically SB- and TPA-treated whole skin. Results normalized to TPA only samples. Reverse transcription–polymerase chain reaction showing induction of these chemokines by TPA (bottom). n = 4–6 mice per treatment group. A, acetone.

    Journal: Carcinogenesis

    Article Title: Use of a TGF? type I receptor inhibitor in mouse skin carcinogenesis reveals a dual role for TGF? signaling in tumor promotion and progression

    doi: 10.1093/carcin/bgq191

    Figure Lengend Snippet: SB suppresses TPA-induced inflammation. ( A ) Effect of SB on chronic TPA-induced hyperplasia (top) proliferation (middle) and apoptosis (bottom) ( B ) Decreased cutaneous neutrophils (MPO+ cells) after acute and chronic TPA + SB treatment. ( C ) Decreased keratinocyte chemokine (KC) and macrophage-inflammatory protein 2 (MIP2) gene expression in acute and chronically SB- and TPA-treated whole skin. Results normalized to TPA only samples. Reverse transcription–polymerase chain reaction showing induction of these chemokines by TPA (bottom). n = 4–6 mice per treatment group. A, acetone.

    Article Snippet: Total RNA was isolated from whole skin using Trizol (Invitrogen, Carlsbad, CA) and quantitative reverse transcription–polymerase chain reaction done for the indicated genes using the MyIQ system (Bio-Rad Laboratories, Hercules, CA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Mouse Assay

    SB rapidly induces progressed phenotype in epidermis-expressing human C-Ha RASV12G oncogene. ( A ) Significant increase in proliferation with SB treatment in RAS -expressing skin (left) and slight increase in epidermal thickness (right). ( B ) An increase in MPO+ cells in the dermis of SB-treated mice expressing the InvtTA × tetO RASV12G transgenes. Arrows indicate neutrophils. Yellow line shows epidermal/dermal junction. Representative micrographs (right), magnification, ×100. Scale bar represents 50 μm. ( C ) Increased messenger RNA expression of neutrophil chemokines s100a8, s100a9 and keratinocyte chemokine with treatment of SB. ( D ) SB causes significant decrease in keratin 1 staining (left) and messenger RNA expression (middle). Representative micrographs (left), magnification ×100. Scale bar represents 50 μm. Yellow dotted line shows epidermal/dermal junction. Significant increase in keratin 18 messenger RNA expression by quantitative reverse transcription–polymerase chain reaction in RAS -expressing skin following SB (right). n = 4–6 mice per treatment group.

    Journal: Carcinogenesis

    Article Title: Use of a TGF? type I receptor inhibitor in mouse skin carcinogenesis reveals a dual role for TGF? signaling in tumor promotion and progression

    doi: 10.1093/carcin/bgq191

    Figure Lengend Snippet: SB rapidly induces progressed phenotype in epidermis-expressing human C-Ha RASV12G oncogene. ( A ) Significant increase in proliferation with SB treatment in RAS -expressing skin (left) and slight increase in epidermal thickness (right). ( B ) An increase in MPO+ cells in the dermis of SB-treated mice expressing the InvtTA × tetO RASV12G transgenes. Arrows indicate neutrophils. Yellow line shows epidermal/dermal junction. Representative micrographs (right), magnification, ×100. Scale bar represents 50 μm. ( C ) Increased messenger RNA expression of neutrophil chemokines s100a8, s100a9 and keratinocyte chemokine with treatment of SB. ( D ) SB causes significant decrease in keratin 1 staining (left) and messenger RNA expression (middle). Representative micrographs (left), magnification ×100. Scale bar represents 50 μm. Yellow dotted line shows epidermal/dermal junction. Significant increase in keratin 18 messenger RNA expression by quantitative reverse transcription–polymerase chain reaction in RAS -expressing skin following SB (right). n = 4–6 mice per treatment group.

    Article Snippet: Total RNA was isolated from whole skin using Trizol (Invitrogen, Carlsbad, CA) and quantitative reverse transcription–polymerase chain reaction done for the indicated genes using the MyIQ system (Bio-Rad Laboratories, Hercules, CA).

    Techniques: Expressing, Mouse Assay, RNA Expression, Staining, Reverse Transcription Polymerase Chain Reaction

    SB reduces TPA-induced Smad2 phosphorylation in vivo . ( A (available at Carcinogenesis Online). ( B ) Quantitative reverse transcription–polymerase chain reaction analysis of Smad7 and LTBP-1 gene expression in acetone or SB-treated whole skin. Normalized to acetone only samples ( n = 4). ( C ) Indirect immunofluorescence showing a reduction in TPA-induced p-Smad2 by SB at 4 and 24 h TPA treatment. Magnification ×800, bar represents 15 μm. P-Smad2 was detected with Alexa-fluor-488 (green), and nuclei counterstained with TO-PRO3 (red), (representative of n (available at Carcinogenesis Online). ( D ) Western blot of whole skin treated 24 h with TPA and 12 h SB, showing reduced TPA-induced Smad2 phosphorylation with SB treatment (representative of n (available at Carcinogenesis Online). C, control and A, acetone.

    Journal: Carcinogenesis

    Article Title: Use of a TGF? type I receptor inhibitor in mouse skin carcinogenesis reveals a dual role for TGF? signaling in tumor promotion and progression

    doi: 10.1093/carcin/bgq191

    Figure Lengend Snippet: SB reduces TPA-induced Smad2 phosphorylation in vivo . ( A (available at Carcinogenesis Online). ( B ) Quantitative reverse transcription–polymerase chain reaction analysis of Smad7 and LTBP-1 gene expression in acetone or SB-treated whole skin. Normalized to acetone only samples ( n = 4). ( C ) Indirect immunofluorescence showing a reduction in TPA-induced p-Smad2 by SB at 4 and 24 h TPA treatment. Magnification ×800, bar represents 15 μm. P-Smad2 was detected with Alexa-fluor-488 (green), and nuclei counterstained with TO-PRO3 (red), (representative of n (available at Carcinogenesis Online). ( D ) Western blot of whole skin treated 24 h with TPA and 12 h SB, showing reduced TPA-induced Smad2 phosphorylation with SB treatment (representative of n (available at Carcinogenesis Online). C, control and A, acetone.

    Article Snippet: Total RNA was isolated from whole skin using Trizol (Invitrogen, Carlsbad, CA) and quantitative reverse transcription–polymerase chain reaction done for the indicated genes using the MyIQ system (Bio-Rad Laboratories, Hercules, CA).

    Techniques: In Vivo, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence, Western Blot

    Ig gene rearrangement and transcription in μMT mice liver epithelial cells. ( A ) Northern blot analysis. Total RNA from liver cells of three μMT mice and WT spleen cells (positive control) were transferred to nylon membrane, and probed with probes to constant region of Igμ and Igκ; 28 s and 18 s RNA were shown by DNA agarose gel electrophoresis. ( B ) In situ hybridization analysis was shown for detection of Ig μ and Ig κ transcripts in μMT mice with antisense or sense probes for C region of μ chain or J-C region of κ chain. ( C ) a It was shown for the primer design of Ig μ variable region, Ig κ variable region and Igλ; b Sorted liver epithelial cells from two μMT mice were analyzed for Ig rearrangement and transcription by RT-PCR. WT spleen cells were used as the positive control. Variable region and constant region of heavy chain (μ, α, δ, γ, ε) and light chain (κ, λ), CD20 and GAPDH were amplified; “1, 2” represented two samples of sorted μMT liver epithelial cells.

    Journal: Scientific Reports

    Article Title: Identification of Liver Epithelial Cell-derived Ig Expression in μ chain-deficient mice

    doi: 10.1038/srep23669

    Figure Lengend Snippet: Ig gene rearrangement and transcription in μMT mice liver epithelial cells. ( A ) Northern blot analysis. Total RNA from liver cells of three μMT mice and WT spleen cells (positive control) were transferred to nylon membrane, and probed with probes to constant region of Igμ and Igκ; 28 s and 18 s RNA were shown by DNA agarose gel electrophoresis. ( B ) In situ hybridization analysis was shown for detection of Ig μ and Ig κ transcripts in μMT mice with antisense or sense probes for C region of μ chain or J-C region of κ chain. ( C ) a It was shown for the primer design of Ig μ variable region, Ig κ variable region and Igλ; b Sorted liver epithelial cells from two μMT mice were analyzed for Ig rearrangement and transcription by RT-PCR. WT spleen cells were used as the positive control. Variable region and constant region of heavy chain (μ, α, δ, γ, ε) and light chain (κ, λ), CD20 and GAPDH were amplified; “1, 2” represented two samples of sorted μMT liver epithelial cells.

    Article Snippet: RNA extraction and RT-PCR Total RNA in spleen was extracted using TRIzol reagent (Invitrogen, Carlsbad, USA).

    Techniques: Mouse Assay, Northern Blot, Positive Control, Agarose Gel Electrophoresis, In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Amplification

    In utero B(a)P-exposure upregulates heart nNOS, eNOS, BH4/BH2 oxidore-ductase and AngII mRNA in offspring. Relative expression levels of nNOS, eNOS, BH4/BH2 and AngII as measured by qRT-PCR. Quantitative RT-PCR was performed using iScript one-step RT-PCR

    Journal: Toxicology

    Article Title: In utero exposure to benzo(a)pyrene predisposes offspring to cardiovascular dysfunction in later-life

    doi: 10.1016/j.tox.2012.01.017

    Figure Lengend Snippet: In utero B(a)P-exposure upregulates heart nNOS, eNOS, BH4/BH2 oxidore-ductase and AngII mRNA in offspring. Relative expression levels of nNOS, eNOS, BH4/BH2 and AngII as measured by qRT-PCR. Quantitative RT-PCR was performed using iScript one-step RT-PCR

    Article Snippet: Quantitative Real time RT-PCR was carried out using the iScript one-step RT-PCR kit with SYBR Green, PCR plates and optically clear adhesive plate seals (Bio-Rad Laboratories, USA).

    Techniques: In Utero, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    Nectin-4 and Nectin-1 are expressed in human mesothelial cells and ovarian cancer patient samples RT-PCR analysis of Nectin-4 and Nectin-1 expression in human mesothelial cell lines LP9 and Met5a, and matched samples from four patients with high grade serous ovarian cancer: ascites cells (As), primary ovarian tumor (Ov), and omental metastases (Om). Nectin-4 RNA was expressed in all of the samples, at variable levels. Nectin-1 RNA was more consistently expressed across samples. β-actin, loading control.

    Journal: Oncotarget

    Article Title: The expression of Nectin-4 on the surface of ovarian cancer cells alters their ability to adhere, migrate, aggregate, and proliferate

    doi: 10.18632/oncotarget.14206

    Figure Lengend Snippet: Nectin-4 and Nectin-1 are expressed in human mesothelial cells and ovarian cancer patient samples RT-PCR analysis of Nectin-4 and Nectin-1 expression in human mesothelial cell lines LP9 and Met5a, and matched samples from four patients with high grade serous ovarian cancer: ascites cells (As), primary ovarian tumor (Ov), and omental metastases (Om). Nectin-4 RNA was expressed in all of the samples, at variable levels. Nectin-1 RNA was more consistently expressed across samples. β-actin, loading control.

    Article Snippet: 50-100 ng of total RNA was amplified using the Access RT-PCR System (Promega Corporation, Madison, WI), as previously described [ ].

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

    Establishment and validation of multivirus real‐time PCR. A : Procedure of the multivirus real‐time PCR system. DNA sample was mixed with Quantitect 2× master mix, and RNA sample was mixed with Quantitect 2× master mix and reverse transcriptase (RT) mix. These mixtures were poured into each well in a 96‐well plate at 10 µl per well. Ten microliters of 2× probe and primer mix were then added to each well in a premixed 96‐well plate. Finally, the virus genes were amplified and detected in a real‐time PCR machine for 2 hr. B : Validation of the multivirus real‐time PCR. A gene expression image by TreeView software based on the results of the multivirus real‐time PCR for control samples is shown. A horizontal line shows each probe–primer set and a vertical line is one sample of positive control. Gray vertical lines indicate no sample. A scale bar indicates copy number of color. A green box indicates specific reactions of target positive controls in specific probe–primer sets. Arrows of (a–c) also show specific signals. The arrow (a) shows positive signal for TTV in a brain sample with both JCV and TTV infection. The arrows (b1–3) show that a probe–primer set for pan‐enterovirus reacted with poliovirus (b1), Coxsackievirus B3 (b2), and Echovirus 6 (b3) positive samples. The arrow (c) shows that a probe–primer set for influenza virus A reacted with H5N1 influenza virus. Details of positive controls were listed in Supplementary Table II.

    Journal: Journal of Medical Virology

    Article Title: A novel real‐time PCR system for simultaneous detection of human viruses in clinical samples from patients with uncertain diagnoses

    doi: 10.1002/jmv.21962

    Figure Lengend Snippet: Establishment and validation of multivirus real‐time PCR. A : Procedure of the multivirus real‐time PCR system. DNA sample was mixed with Quantitect 2× master mix, and RNA sample was mixed with Quantitect 2× master mix and reverse transcriptase (RT) mix. These mixtures were poured into each well in a 96‐well plate at 10 µl per well. Ten microliters of 2× probe and primer mix were then added to each well in a premixed 96‐well plate. Finally, the virus genes were amplified and detected in a real‐time PCR machine for 2 hr. B : Validation of the multivirus real‐time PCR. A gene expression image by TreeView software based on the results of the multivirus real‐time PCR for control samples is shown. A horizontal line shows each probe–primer set and a vertical line is one sample of positive control. Gray vertical lines indicate no sample. A scale bar indicates copy number of color. A green box indicates specific reactions of target positive controls in specific probe–primer sets. Arrows of (a–c) also show specific signals. The arrow (a) shows positive signal for TTV in a brain sample with both JCV and TTV infection. The arrows (b1–3) show that a probe–primer set for pan‐enterovirus reacted with poliovirus (b1), Coxsackievirus B3 (b2), and Echovirus 6 (b3) positive samples. The arrow (c) shows that a probe–primer set for influenza virus A reacted with H5N1 influenza virus. Details of positive controls were listed in Supplementary Table II.

    Article Snippet: In addition, the duplex real‐time PCR using Quantitect Multiplex Probe RT‐PCR kit (Qiagen) had similar sensitivity to single real‐time PCR procedures using Quantitect Probe RT‐PCR kit (Qiagen) in several probe–primer sets (Supplementary Fig. 1B).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Expressing, Software, Positive Control, Infection

    FatB was expressed in B. thuringiensis cells grown in iron restricted medium at 25 °C, 30 °C and 37 °C. RNA was isolated from stationary phase cells and reverse transcription PCR with 35 cycles was used to detect gene expression. Experiments were performed in triplicate and a representative gel is shown.

    Journal: International Journal of Molecular Sciences

    Article Title: Production of Protocatechuic Acid in Bacillus Thuringiensis ATCC33679

    doi: 10.3390/ijms13033765

    Figure Lengend Snippet: FatB was expressed in B. thuringiensis cells grown in iron restricted medium at 25 °C, 30 °C and 37 °C. RNA was isolated from stationary phase cells and reverse transcription PCR with 35 cycles was used to detect gene expression. Experiments were performed in triplicate and a representative gel is shown.

    Article Snippet: RNA was used as a template for Invitrogen SuperScript™ One-Step RT-PCR at a concentration of 5 nangrams.

    Techniques: Isolation, Polymerase Chain Reaction, Expressing

    AsbF was expressed in B. thuringiensis cells grown in iron restricted medium at 25 °C, 30 °C and 37 °C. RNA was isolated from stationary phase cells and reverse transcription PCR with 25 cycles was used to detect gene expression. Experiments were performed in triplicate and a representative gel is shown the internal control tuf .

    Journal: International Journal of Molecular Sciences

    Article Title: Production of Protocatechuic Acid in Bacillus Thuringiensis ATCC33679

    doi: 10.3390/ijms13033765

    Figure Lengend Snippet: AsbF was expressed in B. thuringiensis cells grown in iron restricted medium at 25 °C, 30 °C and 37 °C. RNA was isolated from stationary phase cells and reverse transcription PCR with 25 cycles was used to detect gene expression. Experiments were performed in triplicate and a representative gel is shown the internal control tuf .

    Article Snippet: RNA was used as a template for Invitrogen SuperScript™ One-Step RT-PCR at a concentration of 5 nangrams.

    Techniques: Isolation, Polymerase Chain Reaction, Expressing

    p53-mediated induction of Apaf1 mRNA in neurons. (A) RNA was extracted from neurons 36 or 48 h after infection with Ad-p53 or Ad-p53-173L and analyzed for Apaf1 or GAPDH expression using semiquantitative RT-PCR. (B) RNA was extracted from wild-type or p53-deficient neurons at the indicated times after treatment with 10 μM camptothecin and analyzed for Apaf1 or GAPDH expression using semiquantitative RT-PCR.

    Journal: The Journal of Cell Biology

    Article Title: APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death

    doi: 10.1083/jcb.200105137

    Figure Lengend Snippet: p53-mediated induction of Apaf1 mRNA in neurons. (A) RNA was extracted from neurons 36 or 48 h after infection with Ad-p53 or Ad-p53-173L and analyzed for Apaf1 or GAPDH expression using semiquantitative RT-PCR. (B) RNA was extracted from wild-type or p53-deficient neurons at the indicated times after treatment with 10 μM camptothecin and analyzed for Apaf1 or GAPDH expression using semiquantitative RT-PCR.

    Article Snippet: 2 ng of total RNA was used for cDNA synthesis and targeted gene amplification using the SuperScript One-Step RT-PCR kit (GIBCO BRL). cDNA synthesis was carried out at 48°C for 45 min followed by a 2 min initial denaturation step at 94°C.

    Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction

    Expression analysis of eight Arabidopsis genes. A, Total RNA was isolated from root (R), hypocotyl (H), cotyledon (C), leaf (L), and stem (S) tissues of 3-week-old in vitro-germinated plants and cell suspension cultures (Cs). RT-PCR was performed on equal

    Journal: Plant Physiology

    Article Title: Gene Trapping with Firefly Luciferase in Arabidopsis. Tagging of Stress-Responsive Genes 1Gene Trapping with Firefly Luciferase in Arabidopsis. Tagging of Stress-Responsive Genes 1 [w]

    doi: 10.1104/pp.103.027151

    Figure Lengend Snippet: Expression analysis of eight Arabidopsis genes. A, Total RNA was isolated from root (R), hypocotyl (H), cotyledon (C), leaf (L), and stem (S) tissues of 3-week-old in vitro-germinated plants and cell suspension cultures (Cs). RT-PCR was performed on equal

    Article Snippet: RNA samples were isolated from 3-week-old seedlings as described ( ). cDNA templates were prepared using 1 μg of RNA and an RT-PCR kit (CLONTECH Laboratories, Palo Alto, CA) according to the supplier's instructions.

    Techniques: Expressing, Isolation, In Vitro, Reverse Transcription Polymerase Chain Reaction

    2.8. SYBR green real-time reverse transcription-polymerase chain reaction (RT-PCR)

    Journal: Journal of Zhejiang University. Science. B

    Article Title: Growth factors induce the improved cardiac remodeling in autologous mesenchymal stem cell-implanted failing rat hearts

    doi: 10.1631/jzus.B0900244

    Figure Lengend Snippet: 2.8. SYBR green real-time reverse transcription-polymerase chain reaction (RT-PCR)

    Article Snippet: The SYBR green real-time RT-PCR amplifications were performed using SYBR® PrimeScript™ RT-PCR Kit (TaKaRa Biotech Co.) and Applied Biosystems 7500 System (Applied Biosystems, USA).

    Techniques: SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction

    Compensatory analysis of miR21 knock-down targeted to miR21 primary transcript by snoMEN vector. ( a ) Micrographs showing rescue experiment of apoptosis induction by co-transfecting with pri-miR21-snoMEN (mCherry) and pre-miR21 expression plasmid (GFP+miR21)/Control plasmid that expresses GFP protein alone (GFP). Images were taken 72 hours followed after transfection. Scale bar is 10 μm. ( b ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after co-transfection with pri-miR21-snoMEN (mCherry) and pre-miR21 expression plasmid (miR21)/Control plasmid that express GFP protein alone (GFP) and qRT-PCR was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21, Red). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21, Green). U3 snoRNA was used as a control. Graph depicts mean and standard deviation from a minimum of 3 independent experiments. ( c ) Micrographs show prevention of non-apoptosis induction by transfecting with pri-miR21-snoMEN mut, which has 3 base mismatches in each M box complementary sequence to pri-miR21. Images were taken 72 hours followed after transfection. Scale bar is 10 μm. ( d ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after transfection with either pri-miR21-snoMEN mut or Control. Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ). U3 snoRNA was used as a control. Graph depicts mean and standard deviation from a minimum of 4 independent experiments.

    Journal: PLoS ONE

    Article Title: Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines

    doi: 10.1371/journal.pone.0138668

    Figure Lengend Snippet: Compensatory analysis of miR21 knock-down targeted to miR21 primary transcript by snoMEN vector. ( a ) Micrographs showing rescue experiment of apoptosis induction by co-transfecting with pri-miR21-snoMEN (mCherry) and pre-miR21 expression plasmid (GFP+miR21)/Control plasmid that expresses GFP protein alone (GFP). Images were taken 72 hours followed after transfection. Scale bar is 10 μm. ( b ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after co-transfection with pri-miR21-snoMEN (mCherry) and pre-miR21 expression plasmid (miR21)/Control plasmid that express GFP protein alone (GFP) and qRT-PCR was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21, Red). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21, Green). U3 snoRNA was used as a control. Graph depicts mean and standard deviation from a minimum of 3 independent experiments. ( c ) Micrographs show prevention of non-apoptosis induction by transfecting with pri-miR21-snoMEN mut, which has 3 base mismatches in each M box complementary sequence to pri-miR21. Images were taken 72 hours followed after transfection. Scale bar is 10 μm. ( d ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after transfection with either pri-miR21-snoMEN mut or Control. Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ). U3 snoRNA was used as a control. Graph depicts mean and standard deviation from a minimum of 4 independent experiments.

    Article Snippet: The QuantiFast SYBR green RT-PCR kit (QIAGEN) and PerfeCta SYBR Green qPCR kit (Quanta Biosciences), were used to analyse samples for qRT-PCR and qPCR, respectively, on the LightCycler 480 II platform (Roche).

    Techniques: Plasmid Preparation, Expressing, Transfection, Cotransfection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation, Sequencing

    miR21 knock-down using si/shRNA targeted to endogenous miR21 primary transcript. ( a ) The regions on the pri-miR21 RNA targeted by the snoMEN vector used in this study are shown in a schematic diagram (sh/si21M1-M3). The same pri-miR21 sequences as targeted by the snoMEN vector were targeted by siRNA oligoribonucleotides and shRNA expression plasmids. ( b ) The graph shows the result of qRT-PCR/qPCR. Total RNA from HeLa cells was harvested 24 hours after transfection and qRT-PCR was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21, Red). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21, Green). U3 was used as a control. Graph depicts mean and standard deviation from a minimum of 4 independent experiments. The right panel shows the result of Northern blot analysis for siRNA experiments. Detection of endogenous miR21 RNA levels following transfection of HeLa cells using either control siRNA (Scramble: lane1), miR21 siRNA (si21: lane2), miR21 M box siRNA-1 (si21M1: lane3), miR21 M box siRNA-2 (si21M2: lane4), miR21 M box siRNA-3 (si21M3: lane5). An equivalent amount of HeLa total RNA was loaded for each lane and the RNA separated by PAGE, electroblotted onto membrane and probed both with a miR21 probe and with a tRNA probe as a loading control. ( c ) The same series of experiments with siRNA transfection, as described in Fig 6b , except shRNA expression plasmids were transfected instead of siRNAs.

    Journal: PLoS ONE

    Article Title: Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines

    doi: 10.1371/journal.pone.0138668

    Figure Lengend Snippet: miR21 knock-down using si/shRNA targeted to endogenous miR21 primary transcript. ( a ) The regions on the pri-miR21 RNA targeted by the snoMEN vector used in this study are shown in a schematic diagram (sh/si21M1-M3). The same pri-miR21 sequences as targeted by the snoMEN vector were targeted by siRNA oligoribonucleotides and shRNA expression plasmids. ( b ) The graph shows the result of qRT-PCR/qPCR. Total RNA from HeLa cells was harvested 24 hours after transfection and qRT-PCR was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21, Red). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21, Green). U3 was used as a control. Graph depicts mean and standard deviation from a minimum of 4 independent experiments. The right panel shows the result of Northern blot analysis for siRNA experiments. Detection of endogenous miR21 RNA levels following transfection of HeLa cells using either control siRNA (Scramble: lane1), miR21 siRNA (si21: lane2), miR21 M box siRNA-1 (si21M1: lane3), miR21 M box siRNA-2 (si21M2: lane4), miR21 M box siRNA-3 (si21M3: lane5). An equivalent amount of HeLa total RNA was loaded for each lane and the RNA separated by PAGE, electroblotted onto membrane and probed both with a miR21 probe and with a tRNA probe as a loading control. ( c ) The same series of experiments with siRNA transfection, as described in Fig 6b , except shRNA expression plasmids were transfected instead of siRNAs.

    Article Snippet: The QuantiFast SYBR green RT-PCR kit (QIAGEN) and PerfeCta SYBR Green qPCR kit (Quanta Biosciences), were used to analyse samples for qRT-PCR and qPCR, respectively, on the LightCycler 480 II platform (Roche).

    Techniques: shRNA, Plasmid Preparation, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Transfection, SYBR Green Assay, Standard Deviation, Northern Blot, Polyacrylamide Gel Electrophoresis

    snoMEN vector targeted to miR21 primary transcript. ( a ) Structure for targeted endogenous miR21 primary transcript (mCherry–pri-miR21 snoMEN) and schematic diagram of miR21 maturation pathway. This construct has three snoMEN RNAs (blue pentagons) as previously described[ 1 ], except that here the M box sequences are complementary to specific sequences within the endogenous miR21 primary transcript. ( b ) Validation of snoMEN expression by Fluorescence In Situ Hybridisation (FISH) analysis. Each snoMEN RNA was detected by using a M box specific RNA probe labelled with Cy3 (Cy3). DNA is stained by DAPI (DAPI). Scale bar is 10 μm. ( c ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after transfection and qRT-PCR was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21). U3 was used as a loading control. Graph depicts mean and standard deviation from a minimum of 5 independent experiments. ( d ) HeLa cells transfected with pri-miR21-snoMEN/Control for 24 hours prior to total RNA extraction and northern blot analysis.

    Journal: PLoS ONE

    Article Title: Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines

    doi: 10.1371/journal.pone.0138668

    Figure Lengend Snippet: snoMEN vector targeted to miR21 primary transcript. ( a ) Structure for targeted endogenous miR21 primary transcript (mCherry–pri-miR21 snoMEN) and schematic diagram of miR21 maturation pathway. This construct has three snoMEN RNAs (blue pentagons) as previously described[ 1 ], except that here the M box sequences are complementary to specific sequences within the endogenous miR21 primary transcript. ( b ) Validation of snoMEN expression by Fluorescence In Situ Hybridisation (FISH) analysis. Each snoMEN RNA was detected by using a M box specific RNA probe labelled with Cy3 (Cy3). DNA is stained by DAPI (DAPI). Scale bar is 10 μm. ( c ) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after transfection and qRT-PCR was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ) (Matured-miR21). U3 was used as a loading control. Graph depicts mean and standard deviation from a minimum of 5 independent experiments. ( d ) HeLa cells transfected with pri-miR21-snoMEN/Control for 24 hours prior to total RNA extraction and northern blot analysis.

    Article Snippet: The QuantiFast SYBR green RT-PCR kit (QIAGEN) and PerfeCta SYBR Green qPCR kit (Quanta Biosciences), were used to analyse samples for qRT-PCR and qPCR, respectively, on the LightCycler 480 II platform (Roche).

    Techniques: Plasmid Preparation, Construct, Expressing, Fluorescence, In Situ, Hybridization, Fluorescence In Situ Hybridization, Staining, Transfection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation, RNA Extraction, Northern Blot

    Ago2 is important for snoMEN RNA interference. ( a ) HeLa cells were transfected with siRNA oligonucleotides for Ago1, Ago2, and Upf1. Whole cell lysates were prepared 48 hours after transfections and analysed by western blot using the indicated antibodies. ( b ) HeLa cells were transfected with siRNA oligonucleotides prior to pri-miR21-snoMEN transfection for 24h. Total RNA from HeLa cells was harvested and qPCR was performed, followed by cDNA synthesis, using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ). ( c ) HeLa cells transfected with pri-miR21-snoMEN for 24 hours prior to protein extraction. The same amount of proteins/RNAs from each subcellular fraction was loaded on each lane and analysed by western blot/Northern blot using the indicated antibodies (Ago1, Ago2, Upf1) / RI-labelled probes (snoMEN, tRNA). The fractionation markers, i.e. Tubulin (Cytoplasm), Lamin A/C (Nucleoplasm), Fibrillarin (Nucleoli), were tested to evaluate fractionation quality. ( d ) Images show localisation pattern of DNA (DAPI, Blue), a transfection FP-marker of pri-miR21-snoMEN (mCherry, Red) and the indicated proteins, i.e. Ago1, Ago2 and Upf1 (FITC, Green). Scale bar is 10 μm. Arrows and arrowheads show transfected and non-transfected cells, respectively. Specificity of each antibody was also verified by siRNA transfections ( Fig K in S1 File ). ( e ) HeLa cells were transfected with pri-miR21-snoMEN for 24 hours prior to fractionation. Cell lysates of each subcellular fraction were prepared and immunoprecipitated with either normal mouse IgG, or anti-Ago2 antibodies. Total protein was isolated from precipitates and resolved by SDS-PAGE and then analysed by western blotting using an Ago2 antibody to validate the efficiency of purification. In parallel, total RNA was also isolated from each precipitate and qRT-PCR was performed using the specific primers indicated.

    Journal: PLoS ONE

    Article Title: Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines

    doi: 10.1371/journal.pone.0138668

    Figure Lengend Snippet: Ago2 is important for snoMEN RNA interference. ( a ) HeLa cells were transfected with siRNA oligonucleotides for Ago1, Ago2, and Upf1. Whole cell lysates were prepared 48 hours after transfections and analysed by western blot using the indicated antibodies. ( b ) HeLa cells were transfected with siRNA oligonucleotides prior to pri-miR21-snoMEN transfection for 24h. Total RNA from HeLa cells was harvested and qPCR was performed, followed by cDNA synthesis, using matured miR21 specific primer and universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods ). ( c ) HeLa cells transfected with pri-miR21-snoMEN for 24 hours prior to protein extraction. The same amount of proteins/RNAs from each subcellular fraction was loaded on each lane and analysed by western blot/Northern blot using the indicated antibodies (Ago1, Ago2, Upf1) / RI-labelled probes (snoMEN, tRNA). The fractionation markers, i.e. Tubulin (Cytoplasm), Lamin A/C (Nucleoplasm), Fibrillarin (Nucleoli), were tested to evaluate fractionation quality. ( d ) Images show localisation pattern of DNA (DAPI, Blue), a transfection FP-marker of pri-miR21-snoMEN (mCherry, Red) and the indicated proteins, i.e. Ago1, Ago2 and Upf1 (FITC, Green). Scale bar is 10 μm. Arrows and arrowheads show transfected and non-transfected cells, respectively. Specificity of each antibody was also verified by siRNA transfections ( Fig K in S1 File ). ( e ) HeLa cells were transfected with pri-miR21-snoMEN for 24 hours prior to fractionation. Cell lysates of each subcellular fraction were prepared and immunoprecipitated with either normal mouse IgG, or anti-Ago2 antibodies. Total protein was isolated from precipitates and resolved by SDS-PAGE and then analysed by western blotting using an Ago2 antibody to validate the efficiency of purification. In parallel, total RNA was also isolated from each precipitate and qRT-PCR was performed using the specific primers indicated.

    Article Snippet: The QuantiFast SYBR green RT-PCR kit (QIAGEN) and PerfeCta SYBR Green qPCR kit (Quanta Biosciences), were used to analyse samples for qRT-PCR and qPCR, respectively, on the LightCycler 480 II platform (Roche).

    Techniques: Transfection, Western Blot, Real-time Polymerase Chain Reaction, SYBR Green Assay, Protein Extraction, Northern Blot, Fractionation, Marker, Immunoprecipitation, Isolation, SDS Page, Purification, Quantitative RT-PCR