rt-pcr kit Search Results


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  • 99
    Thermo Fisher rt pcr kit
    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 <t>qRT-PCR</t> 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.
    Rt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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 14902 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    TaKaRa primescript 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.
    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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    94
    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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    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.
    Quantitect Probe Rt Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 3045 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
    Rt Pcr Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 3157 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript one step rt pcr kit
    p53-mediated induction of Apaf1 mRNA in neurons. (A) <t>RNA</t> 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 <t>RT-PCR.</t> (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.
    Superscript One Step Rt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2504 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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.
    Quantifast Sybr Green Rt Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2615 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher agpath id one step rt pcr kit
    Effect of enzyme system on detection of pathogen targets in primary clinical specimens. Data shown are difference in Ct value between reactions using Quanta One-step <t>RT-PCR</t> ToughMix and <t>AgPath-ID</t> One-step RT-PCR kit when testing TNA extracted from NP/OP swabs (A) or blood (B). Each data point represents the difference in Ct value between the two reactions for an individual clinical specimen. Median difference is indicated ( ― ) for assays with ≥2 positive results. *Targets that were only detected using AgPath always occurred when Ct values were > 33.
    Agpath Id One Step Rt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1859 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii one step rt pcr kit
    Full-length AKAP13 mRNA levels are reduced by the gene-trap events. (A) TaqMan gene expression assays were used to measure the expression of AKAP13 transcripts at the indicated exon-exon junctions (E4-5, Brx-9, E37-38). (B) Quantitative <t>PCR</t> analysis of wild-type (WT), heterozygote (Het) and homozygote (Hom) neonatal mouse heart and lung RNA for AKAP13 showed that none of the gene-trap mutations affected expression of the E4-5 exon-exon junction. The ΔBrx gene-trap dose dependently decreased expression of the Brx-9 exon-exon junction. Expression of the Brx-9 junction was eliminated in the AKAP13 ΔBrx/ΔBrx mice. All <t>three</t> gene-traps decreased expression of the E37-38 exon-exon junction in a dose-dependent manner. Expression of the E37-38 junction was eliminated in the AKAP13 ΔGEF/ΔGEF mice. The means and standard deviations are graphed for six mice per genotype. One-way ANOVA and Bonferroni’s multiple comparison tests were conducted (Prism 5; GraphPad). †, p
    Superscript Iii One Step Rt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1761 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher one step rt pcr kit
    Expression of TANK1 and TANK1a in WT and TANK1 −/− mouse tissues. All <t>RNA</t> samples were serially diluted as indicated for <t>PCR</t> amplification and analysis. Actin cDNA was used as an RT-PCR loading control in all experiments. (A) RT-PCR analysis for TANK1 mRNA expression in various tissues of WT and TANK1 −/− (KO) mice as indicated. 5′-TANK1 RT-PCR products represent upstream cDNA of TANK1. (B) RT-PCR analysis for TANK1 mRNA expression in WT and TANK1 −/− (KO) testis as indicated. 5′-TANK1 and 3′-TANK1 RT-PCR products represent upstream and downstream cDNAs of TANK1; and TANK1a RT-PCR products are specific for TANK1a. (C) RT-PCR analysis for TANK1a mRNA expression in various tissues of WT mice as indicated. TANK1a RT-PCR products represent cDNA of TANK1a. (D, E) Western blot analysis was used to determine tankyrase 1 and 1a expression in thymus, testis and spleen of WT and TANK1 −/− (KO) mice with 762 (anti-SAM, D) and 376 (anti-HPS, E) antibodies. TANK1 indicates tankyrase 1 protein, TANK1x indicates a possible degraded tankyrase 1 protein, and TANK1a indicates tankyrase 1a protein.
    One Step Rt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1466 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa one step rt pcr kit
    PGRN deficiency increased the production of systemic and local pro‐inflammatory mediators after injection of LPS . ELISA of serum levels of inflammatory mediators including tumour necrosis factor α ( TNF ‐α) ( A ), interleukin 6 ( IL ‐6) ( B ), IL ‐10 ( C ) and monocyte chemoattractant protein 1 ( MCP ‐1) ( D ). ( E ) Western blot analysis of cold‐inducible <t>RNA</t> ‐binding protein ( CIRP ) and high mobility group box protein 1 ( HMGB 1) in the serum from WT and Grn −/− mice with LPS injection at 6 and 24 hrs. PS red, Ponceau S red staining. Real‐time RT ‐ <t>PCR</t> analysis of mRNA levels of TNF ‐α ( F ), IL ‐6 ( G ), IL ‐1β ( H ) and IL ‐10 ( I ) in the lung after LPS injection. Data are mean ± S.D. * P
    One Step Rt Pcr Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1745 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega access rt pcr kit
    <t>RT-PCR</t> analysis of C. perfringens strains. (A) Total <t>RNA</t> prepared from each specified strain was subjected to RT-PCR analysis using cpb2 -specific internal primers as described in Materials and Methods. RT (+) and RT (−) indicate the presence
    Access Rt Pcr Kit, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1032 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher vetmax plus one step rt pcr kit
    <t>RT-PCR</t> analysis of C. perfringens strains. (A) Total <t>RNA</t> prepared from each specified strain was subjected to RT-PCR analysis using cpb2 -specific internal primers as described in Materials and Methods. RT (+) and RT (−) indicate the presence
    Vetmax Plus One Step Rt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa primescript one step rt pcr kit
    <t>RT-PCR</t> analysis of C. perfringens strains. (A) Total <t>RNA</t> prepared from each specified strain was subjected to RT-PCR analysis using cpb2 -specific internal primers as described in Materials and Methods. RT (+) and RT (−) indicate the presence
    Primescript One Step Rt Pcr Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1014 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    One-step RT-PCR for identification of contaminants in Kit I and Platinum Taq . (A-C) One-step RT-PCR for identification of a contaminated component in Kit I. The experiments were conducted in two independent laboratories, IVR and JRC. In IVR, nucleic acids were extracted from 50 μl of the enzyme mix of the RT-PCR Kit I using an RNA purification column (QIAamp viral RNA mini kit [Cat. no. 52904] [QIAGEN]) and the presence of polytropic endogenous MLV was examined by using the RT-PCR Kit T (A) and Kit P (B). In JRC, nucleic acids were extracted from 75 μl of the enzyme mix of RT-PCR Kit I using an RNA/DNA purification column (PureLink™ Viral RNA/DNA Kit [Cat. no. 12280-050] [Invitrogen]), and the presence of polytropic endogenous MLV was examined using Kit Q (C). Five μl of test samples were examined with primers indicated below the corresponding lanes. The RT-PCR conditions for Kit T and Kit P were the same as in Figure 1B. The RT-PCR conditions for Kit Q were as follows: reverse transcription at 50°C for 30 minutes; activation at 95°C for 15 minutes; 45 cycles of the following steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. Lanes 1 and 5, DW; lanes 2 and 6, column-purified carrier RNA (carrier); lanes 3 and 7, column-purified nucleic acids from enzyme mix (enzyme) of the Kit I; lanes 4 and 8, 1 μl buffer of the Kit I plus 4 μl DW (buffer). (D) One-step RT-PCR for the detection of MLV RNA in Platinum Taq. Nucleic acids were extracted from 50 μl of the Platinum Taq using an RNA purification column (QIAamp viral RNA mini kit [QIAGEN]) and the presence of MLV RNA was examined by using the RT-PCR Kit P. Five μl of test samples were examined with primers indicated below the corresponding lanes. The RT-PCR condition was the same as in Figure 1B with the exception of the PCR cycles (60 cycles instead of 45 cycles). Abbreviation; DW: distilled water. M: DNA size marker.

    Journal: Retrovirology

    Article Title: An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV

    doi: 10.1186/1742-4690-7-110

    Figure Lengend Snippet: One-step RT-PCR for identification of contaminants in Kit I and Platinum Taq . (A-C) One-step RT-PCR for identification of a contaminated component in Kit I. The experiments were conducted in two independent laboratories, IVR and JRC. In IVR, nucleic acids were extracted from 50 μl of the enzyme mix of the RT-PCR Kit I using an RNA purification column (QIAamp viral RNA mini kit [Cat. no. 52904] [QIAGEN]) and the presence of polytropic endogenous MLV was examined by using the RT-PCR Kit T (A) and Kit P (B). In JRC, nucleic acids were extracted from 75 μl of the enzyme mix of RT-PCR Kit I using an RNA/DNA purification column (PureLink™ Viral RNA/DNA Kit [Cat. no. 12280-050] [Invitrogen]), and the presence of polytropic endogenous MLV was examined using Kit Q (C). Five μl of test samples were examined with primers indicated below the corresponding lanes. The RT-PCR conditions for Kit T and Kit P were the same as in Figure 1B. The RT-PCR conditions for Kit Q were as follows: reverse transcription at 50°C for 30 minutes; activation at 95°C for 15 minutes; 45 cycles of the following steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. Lanes 1 and 5, DW; lanes 2 and 6, column-purified carrier RNA (carrier); lanes 3 and 7, column-purified nucleic acids from enzyme mix (enzyme) of the Kit I; lanes 4 and 8, 1 μl buffer of the Kit I plus 4 μl DW (buffer). (D) One-step RT-PCR for the detection of MLV RNA in Platinum Taq. Nucleic acids were extracted from 50 μl of the Platinum Taq using an RNA purification column (QIAamp viral RNA mini kit [QIAGEN]) and the presence of MLV RNA was examined by using the RT-PCR Kit P. Five μl of test samples were examined with primers indicated below the corresponding lanes. The RT-PCR condition was the same as in Figure 1B with the exception of the PCR cycles (60 cycles instead of 45 cycles). Abbreviation; DW: distilled water. M: DNA size marker.

    Article Snippet: We used the following RT-PCR kits which were purchased in Japan: SuperScript® III One-Step RT-PCR System with the Platinum® Taq High Fidelity Kit (Cat. no. 12574-030) (Invitrogen, Carlsbad, CA, USA) (abbreviated as Kit I); AccessQuick™RT-PCR Sysytem (Cat. no. A1701) (Promega, Madison, WI, USA) (abbreviated as Kit P); One Step RT-PCR Kit (Cat. no. PRO24A) (TaKaRa, Ohtsu, Shiga, Japan) (Abbreviated as Kit T); One Step RT-PCR Kit (Cat. no. 210210) (QIAGEN GmbH, Hilden, Germany) (Abbreviated as Kit Q).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Purification, DNA Purification, Activation Assay, Polymerase Chain Reaction, Marker

    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

    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

    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

    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

    Melting peaks of the SYBR Green Ⅰ-based one-step qRT-PCR products. ( A ) Melting peaks of the qRT-PCR products obtained from in vitro transcribed HTNV cRNA ranging from 1×10 8 to 1×10 3 copies/µl. Only a single sharp peak at 84°C was visible in the melt peak chart. ( B ) Melting peaks obtained from serum samples of HFRS patients. All melting curves in A and B have similar shapes.

    Journal: PLoS ONE

    Article Title: Quantification of Hantaan Virus with a SYBR Green I-Based One-Step qRT-PCR Assay

    doi: 10.1371/journal.pone.0081525

    Figure Lengend Snippet: Melting peaks of the SYBR Green Ⅰ-based one-step qRT-PCR products. ( A ) Melting peaks of the qRT-PCR products obtained from in vitro transcribed HTNV cRNA ranging from 1×10 8 to 1×10 3 copies/µl. Only a single sharp peak at 84°C was visible in the melt peak chart. ( B ) Melting peaks obtained from serum samples of HFRS patients. All melting curves in A and B have similar shapes.

    Article Snippet: SYBR Green Ⅰ-based one-step qRT-PCR assay qRT-PCR amplification was performed using a Bio-Rad iQ5 Multi-color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the One-Step SYBR PrimeScript RT-PCR Kit ii (TaKaRa Biotechnology, Dalian, China).

    Techniques: SYBR Green Assay, Quantitative RT-PCR, In Vitro

    Sensitivity of the SYBR Green Ⅰ-based one-step qRT-PCR assay. ( A ) Serial dilutions of the HTNV cRNA standards ranging from 1×10 6 to 1×10 -1 copies/µl were amplified using the SYBR Green Ⅰ-based one-step qRT-PCR assay in duplicate, and the minimum detection limit of the qRT-PCR assay was 10 copies/µl. ( B ) The qRT-PCR products were electrophoresed on a 2% agarose gel. Marker: DNA ladder of DL2000.

    Journal: PLoS ONE

    Article Title: Quantification of Hantaan Virus with a SYBR Green I-Based One-Step qRT-PCR Assay

    doi: 10.1371/journal.pone.0081525

    Figure Lengend Snippet: Sensitivity of the SYBR Green Ⅰ-based one-step qRT-PCR assay. ( A ) Serial dilutions of the HTNV cRNA standards ranging from 1×10 6 to 1×10 -1 copies/µl were amplified using the SYBR Green Ⅰ-based one-step qRT-PCR assay in duplicate, and the minimum detection limit of the qRT-PCR assay was 10 copies/µl. ( B ) The qRT-PCR products were electrophoresed on a 2% agarose gel. Marker: DNA ladder of DL2000.

    Article Snippet: SYBR Green Ⅰ-based one-step qRT-PCR assay qRT-PCR amplification was performed using a Bio-Rad iQ5 Multi-color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the One-Step SYBR PrimeScript RT-PCR Kit ii (TaKaRa Biotechnology, Dalian, China).

    Techniques: SYBR Green Assay, Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis, Marker

    Amplification chart and melt peak chart of HTNV and SEOV. ( A ) Different dilutions of HTNV and SEOV RNA were tested using the SYBR Green Ⅰ-based one-step qRT-PCR assay, and only HTNV RNA was amplified. ( B ) A single sharp peak at 84°C in the melt peak chart indicated that only specific products of HTNV was obtained.

    Journal: PLoS ONE

    Article Title: Quantification of Hantaan Virus with a SYBR Green I-Based One-Step qRT-PCR Assay

    doi: 10.1371/journal.pone.0081525

    Figure Lengend Snippet: Amplification chart and melt peak chart of HTNV and SEOV. ( A ) Different dilutions of HTNV and SEOV RNA were tested using the SYBR Green Ⅰ-based one-step qRT-PCR assay, and only HTNV RNA was amplified. ( B ) A single sharp peak at 84°C in the melt peak chart indicated that only specific products of HTNV was obtained.

    Article Snippet: SYBR Green Ⅰ-based one-step qRT-PCR assay qRT-PCR amplification was performed using a Bio-Rad iQ5 Multi-color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the One-Step SYBR PrimeScript RT-PCR Kit ii (TaKaRa Biotechnology, Dalian, China).

    Techniques: Amplification, SYBR Green Assay, Quantitative RT-PCR

    Expression profiles of ALDH3 family members in human tissues. cDNAs from human liver, kidney, and brain tissue (human MTC (multiple tissue cDNA) panels, Takara Bio) ( A ) or prepared from primary keratinocytes (undifferentiated or differentiated for 7 days; CELLnTEC) ( B ) were subjected to SYBR Green-based real-time quantitative PCR using specific primers for ALDH3A1 , ALDH3A2 , ALDH3B1 , and GAPDH . Values represent the means ± S.D. from three independent reactions. A statistically significant difference is indicated (**, p

    Journal: The Journal of Biological Chemistry

    Article Title: Disruption of the Sjögren-Larsson Syndrome Gene Aldh3a2 in Mice Increases Keratinocyte Growth and Retards Skin Barrier Recovery *

    doi: 10.1074/jbc.M116.714030

    Figure Lengend Snippet: Expression profiles of ALDH3 family members in human tissues. cDNAs from human liver, kidney, and brain tissue (human MTC (multiple tissue cDNA) panels, Takara Bio) ( A ) or prepared from primary keratinocytes (undifferentiated or differentiated for 7 days; CELLnTEC) ( B ) were subjected to SYBR Green-based real-time quantitative PCR using specific primers for ALDH3A1 , ALDH3A2 , ALDH3B1 , and GAPDH . Values represent the means ± S.D. from three independent reactions. A statistically significant difference is indicated (**, p

    Article Snippet: Real-time quantitative PCR was performed using the One-Step SYBR PrimeScript RT-PCR Kit II (Takara Bio) on a CFX96 Touch real-time PCR detection system (Bio-Rad) according to the manufacturer's manual.

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

    Enhanced proliferation and oxidative stress response in Aldh3a2 −/− keratinocytes. A , primary keratinocytes were prepared from wild-type and Aldh3a2 KO mice and grown. Cell numbers were counted by microscopic observation. For each dish, cell numbers in three randomly chosen viewing fields were counted and summed. Values represent the means ± S.D. from four independent experiments. B , keratinocytes prepared from wild-type and Aldh3a2 KO mice were subjected to a [ 3 H]thymidine uptake assay. Values represent the means ± S.D. from three independent experiments. C and D , total RNAs prepared from wild-type and Aldh3a2 KO keratinocytes were subjected to SYBR Green-based real-time quantitative RT-PCR using specific primers for Ki67 ( C ), Hmox1 , Sod1 , Gclc , Gclm , Gsta1 , and Gapdh ( D ). Values are the amount of each mRNA relative to that of Gapdh and represent the means ± S.D. for three independent experiments. Statistically significant differences are indicated (**, p

    Journal: The Journal of Biological Chemistry

    Article Title: Disruption of the Sjögren-Larsson Syndrome Gene Aldh3a2 in Mice Increases Keratinocyte Growth and Retards Skin Barrier Recovery *

    doi: 10.1074/jbc.M116.714030

    Figure Lengend Snippet: Enhanced proliferation and oxidative stress response in Aldh3a2 −/− keratinocytes. A , primary keratinocytes were prepared from wild-type and Aldh3a2 KO mice and grown. Cell numbers were counted by microscopic observation. For each dish, cell numbers in three randomly chosen viewing fields were counted and summed. Values represent the means ± S.D. from four independent experiments. B , keratinocytes prepared from wild-type and Aldh3a2 KO mice were subjected to a [ 3 H]thymidine uptake assay. Values represent the means ± S.D. from three independent experiments. C and D , total RNAs prepared from wild-type and Aldh3a2 KO keratinocytes were subjected to SYBR Green-based real-time quantitative RT-PCR using specific primers for Ki67 ( C ), Hmox1 , Sod1 , Gclc , Gclm , Gsta1 , and Gapdh ( D ). Values are the amount of each mRNA relative to that of Gapdh and represent the means ± S.D. for three independent experiments. Statistically significant differences are indicated (**, p

    Article Snippet: Real-time quantitative PCR was performed using the One-Step SYBR PrimeScript RT-PCR Kit II (Takara Bio) on a CFX96 Touch real-time PCR detection system (Bio-Rad) according to the manufacturer's manual.

    Techniques: Mouse Assay, SYBR Green Assay, Quantitative RT-PCR

    Expression profiles of Aldh3 family members in various tissues. Total RNAs prepared from liver, kidney, retina, cornea, brain, small intestine, lung, testis, spleen, dermis, and epidermis tissue ( A and C ) and from keratinocytes kept undifferentiated or differentiated for 4 days ( B and D ) obtained from wild-type ( A–D ) and Aldh3a2 KO mice ( C and D ) were subjected to SYBR Green-based real-time quantitative RT-PCR using specific primers for Aldh3a1 , Aldh3a2 , Aldh3b1 , Aldh3b2 , Aldh3b3 , and Gapdh . Values are the amount of each mRNA relative to that of Gapdh , and represent the means ± S.D. for three independent experiments. A statistically significant difference is indicated (**, p

    Journal: The Journal of Biological Chemistry

    Article Title: Disruption of the Sjögren-Larsson Syndrome Gene Aldh3a2 in Mice Increases Keratinocyte Growth and Retards Skin Barrier Recovery *

    doi: 10.1074/jbc.M116.714030

    Figure Lengend Snippet: Expression profiles of Aldh3 family members in various tissues. Total RNAs prepared from liver, kidney, retina, cornea, brain, small intestine, lung, testis, spleen, dermis, and epidermis tissue ( A and C ) and from keratinocytes kept undifferentiated or differentiated for 4 days ( B and D ) obtained from wild-type ( A–D ) and Aldh3a2 KO mice ( C and D ) were subjected to SYBR Green-based real-time quantitative RT-PCR using specific primers for Aldh3a1 , Aldh3a2 , Aldh3b1 , Aldh3b2 , Aldh3b3 , and Gapdh . Values are the amount of each mRNA relative to that of Gapdh , and represent the means ± S.D. for three independent experiments. A statistically significant difference is indicated (**, p

    Article Snippet: Real-time quantitative PCR was performed using the One-Step SYBR PrimeScript RT-PCR Kit II (Takara Bio) on a CFX96 Touch real-time PCR detection system (Bio-Rad) according to the manufacturer's manual.

    Techniques: Expressing, Mouse Assay, SYBR Green Assay, Quantitative RT-PCR

    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

    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

    Effect of enzyme system on detection of pathogen targets in primary clinical specimens. Data shown are difference in Ct value between reactions using Quanta One-step RT-PCR ToughMix and AgPath-ID One-step RT-PCR kit when testing TNA extracted from NP/OP swabs (A) or blood (B). Each data point represents the difference in Ct value between the two reactions for an individual clinical specimen. Median difference is indicated ( ― ) for assays with ≥2 positive results. *Targets that were only detected using AgPath always occurred when Ct values were > 33.

    Journal: PLoS ONE

    Article Title: Optimization of Multiple Pathogen Detection Using the TaqMan Array Card: Application for a Population-Based Study of Neonatal Infection

    doi: 10.1371/journal.pone.0066183

    Figure Lengend Snippet: Effect of enzyme system on detection of pathogen targets in primary clinical specimens. Data shown are difference in Ct value between reactions using Quanta One-step RT-PCR ToughMix and AgPath-ID One-step RT-PCR kit when testing TNA extracted from NP/OP swabs (A) or blood (B). Each data point represents the difference in Ct value between the two reactions for an individual clinical specimen. Median difference is indicated ( ― ) for assays with ≥2 positive results. *Targets that were only detected using AgPath always occurred when Ct values were > 33.

    Article Snippet: We compared the performance of two enzyme formulations, Ambion AgPath-ID One-step RT-PCR kit (Applied Biosystems) and Quanta qScript XLT One-step RT-qPCR ToughMix, low ROX (Quanta Biosciences), at detecting targets in NP/OP specimens (n = 18) and blood specimens (n = 12) ( ).

    Techniques: Reverse Transcription Polymerase Chain Reaction

    Full-length AKAP13 mRNA levels are reduced by the gene-trap events. (A) TaqMan gene expression assays were used to measure the expression of AKAP13 transcripts at the indicated exon-exon junctions (E4-5, Brx-9, E37-38). (B) Quantitative PCR analysis of wild-type (WT), heterozygote (Het) and homozygote (Hom) neonatal mouse heart and lung RNA for AKAP13 showed that none of the gene-trap mutations affected expression of the E4-5 exon-exon junction. The ΔBrx gene-trap dose dependently decreased expression of the Brx-9 exon-exon junction. Expression of the Brx-9 junction was eliminated in the AKAP13 ΔBrx/ΔBrx mice. All three gene-traps decreased expression of the E37-38 exon-exon junction in a dose-dependent manner. Expression of the E37-38 junction was eliminated in the AKAP13 ΔGEF/ΔGEF mice. The means and standard deviations are graphed for six mice per genotype. One-way ANOVA and Bonferroni’s multiple comparison tests were conducted (Prism 5; GraphPad). †, p

    Journal: PLoS ONE

    Article Title: AKAP13 Rho-GEF and PKD-Binding Domain Deficient Mice Develop Normally but Have an Abnormal Response to ?-Adrenergic-Induced Cardiac Hypertrophy

    doi: 10.1371/journal.pone.0062705

    Figure Lengend Snippet: Full-length AKAP13 mRNA levels are reduced by the gene-trap events. (A) TaqMan gene expression assays were used to measure the expression of AKAP13 transcripts at the indicated exon-exon junctions (E4-5, Brx-9, E37-38). (B) Quantitative PCR analysis of wild-type (WT), heterozygote (Het) and homozygote (Hom) neonatal mouse heart and lung RNA for AKAP13 showed that none of the gene-trap mutations affected expression of the E4-5 exon-exon junction. The ΔBrx gene-trap dose dependently decreased expression of the Brx-9 exon-exon junction. Expression of the Brx-9 junction was eliminated in the AKAP13 ΔBrx/ΔBrx mice. All three gene-traps decreased expression of the E37-38 exon-exon junction in a dose-dependent manner. Expression of the E37-38 junction was eliminated in the AKAP13 ΔGEF/ΔGEF mice. The means and standard deviations are graphed for six mice per genotype. One-way ANOVA and Bonferroni’s multiple comparison tests were conducted (Prism 5; GraphPad). †, p

    Article Snippet: Total RNA was extracted from ES cells with Trizol (Invitrogen), and RT-PCR was conducted using the SuperScript III One-Step RT-PCR kit (Invitrogen).

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

    Gene-traps disrupt AKAP13 in multiple locations. (A) Schematic of the AKAP13 genomic locus. Exons are depicted with black bars, cassette exons with a grey box, and alternative promoters with arrows. The three gene-trap insertions are indicated. (B) Diagram of the gene-trap constructs (blue boxes) integrated between AKAP13 exons (open boxes with exon numbers). The gene-trap vector contains a strong splice acceptor (SA), βGeo cassette (β−galactosidase and neomyocin resistance genes), and stop codon, as well as a polyadenylation (pA) sequence. The splicing events indicated were confirmed by RT-PCR and sequencing. Primers used to genotype the wild-type and gene-trap alleles are shown (black arrows). (C) Resulting protein fusions of AKAP-Lbc, Brx, and Lbc isoforms with βGeo for the gene-trap mutational series. PKA = protein kinase A binding domain, GEF = Rho-guanine nucleotide exchange factor domain, PKD = protein kinase D binding domain, LZ = leucine zipper domain. (D) Sample genotyping of mouse tail clips for the AKAP13 gene-trap mutations using primers in (B). WT = Wild-type, Het = Heterozygote, Hom = Homozygote.

    Journal: PLoS ONE

    Article Title: AKAP13 Rho-GEF and PKD-Binding Domain Deficient Mice Develop Normally but Have an Abnormal Response to ?-Adrenergic-Induced Cardiac Hypertrophy

    doi: 10.1371/journal.pone.0062705

    Figure Lengend Snippet: Gene-traps disrupt AKAP13 in multiple locations. (A) Schematic of the AKAP13 genomic locus. Exons are depicted with black bars, cassette exons with a grey box, and alternative promoters with arrows. The three gene-trap insertions are indicated. (B) Diagram of the gene-trap constructs (blue boxes) integrated between AKAP13 exons (open boxes with exon numbers). The gene-trap vector contains a strong splice acceptor (SA), βGeo cassette (β−galactosidase and neomyocin resistance genes), and stop codon, as well as a polyadenylation (pA) sequence. The splicing events indicated were confirmed by RT-PCR and sequencing. Primers used to genotype the wild-type and gene-trap alleles are shown (black arrows). (C) Resulting protein fusions of AKAP-Lbc, Brx, and Lbc isoforms with βGeo for the gene-trap mutational series. PKA = protein kinase A binding domain, GEF = Rho-guanine nucleotide exchange factor domain, PKD = protein kinase D binding domain, LZ = leucine zipper domain. (D) Sample genotyping of mouse tail clips for the AKAP13 gene-trap mutations using primers in (B). WT = Wild-type, Het = Heterozygote, Hom = Homozygote.

    Article Snippet: Total RNA was extracted from ES cells with Trizol (Invitrogen), and RT-PCR was conducted using the SuperScript III One-Step RT-PCR kit (Invitrogen).

    Techniques: Construct, Plasmid Preparation, Sequencing, Reverse Transcription Polymerase Chain Reaction, Binding Assay

    EAK-4 Amino Acid Sequence and Alignment with Four C. elegans Homologs Sequences represent the entire predicted amino acid sequences of all five genes. EAK-4 sequence was derived from full-length cDNA amplified by RT-PCR from wild-type C. elegans total RNA. Alignment was constructed using ClustalX 1.8 and MacBoxshade 2.15. Shaded residues indicate amino acid identity and conservation. Mutated residues in three alleles of eak-4 are boxed, and the predicted amino acid change is indicated above the box. The conserved G at residue 2 and S/T/A (boxed) at residue 6 in the N -myristoylation consensus sequence [ 70 ] are denoted by an asterisk and a vertical arrow, respectively.

    Journal: PLoS Genetics

    Article Title: Two Membrane-Associated Tyrosine Phosphatase Homologs Potentiate C. elegans AKT-1/PKB Signaling

    doi: 10.1371/journal.pgen.0020099

    Figure Lengend Snippet: EAK-4 Amino Acid Sequence and Alignment with Four C. elegans Homologs Sequences represent the entire predicted amino acid sequences of all five genes. EAK-4 sequence was derived from full-length cDNA amplified by RT-PCR from wild-type C. elegans total RNA. Alignment was constructed using ClustalX 1.8 and MacBoxshade 2.15. Shaded residues indicate amino acid identity and conservation. Mutated residues in three alleles of eak-4 are boxed, and the predicted amino acid change is indicated above the box. The conserved G at residue 2 and S/T/A (boxed) at residue 6 in the N -myristoylation consensus sequence [ 70 ] are denoted by an asterisk and a vertical arrow, respectively.

    Article Snippet: C. elegans total RNA was isolated as described [ ]. cDNA was amplified from total RNA using the SuperScript III RT-PCR Kit (Invitrogen).

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

    Expression of TANK1 and TANK1a in WT and TANK1 −/− mouse tissues. All RNA samples were serially diluted as indicated for PCR amplification and analysis. Actin cDNA was used as an RT-PCR loading control in all experiments. (A) RT-PCR analysis for TANK1 mRNA expression in various tissues of WT and TANK1 −/− (KO) mice as indicated. 5′-TANK1 RT-PCR products represent upstream cDNA of TANK1. (B) RT-PCR analysis for TANK1 mRNA expression in WT and TANK1 −/− (KO) testis as indicated. 5′-TANK1 and 3′-TANK1 RT-PCR products represent upstream and downstream cDNAs of TANK1; and TANK1a RT-PCR products are specific for TANK1a. (C) RT-PCR analysis for TANK1a mRNA expression in various tissues of WT mice as indicated. TANK1a RT-PCR products represent cDNA of TANK1a. (D, E) Western blot analysis was used to determine tankyrase 1 and 1a expression in thymus, testis and spleen of WT and TANK1 −/− (KO) mice with 762 (anti-SAM, D) and 376 (anti-HPS, E) antibodies. TANK1 indicates tankyrase 1 protein, TANK1x indicates a possible degraded tankyrase 1 protein, and TANK1a indicates tankyrase 1a protein.

    Journal: PLoS ONE

    Article Title: Tankyrase 1 and Tankyrase 2 Are Essential but Redundant for Mouse Embryonic Development

    doi: 10.1371/journal.pone.0002639

    Figure Lengend Snippet: Expression of TANK1 and TANK1a in WT and TANK1 −/− mouse tissues. All RNA samples were serially diluted as indicated for PCR amplification and analysis. Actin cDNA was used as an RT-PCR loading control in all experiments. (A) RT-PCR analysis for TANK1 mRNA expression in various tissues of WT and TANK1 −/− (KO) mice as indicated. 5′-TANK1 RT-PCR products represent upstream cDNA of TANK1. (B) RT-PCR analysis for TANK1 mRNA expression in WT and TANK1 −/− (KO) testis as indicated. 5′-TANK1 and 3′-TANK1 RT-PCR products represent upstream and downstream cDNAs of TANK1; and TANK1a RT-PCR products are specific for TANK1a. (C) RT-PCR analysis for TANK1a mRNA expression in various tissues of WT mice as indicated. TANK1a RT-PCR products represent cDNA of TANK1a. (D, E) Western blot analysis was used to determine tankyrase 1 and 1a expression in thymus, testis and spleen of WT and TANK1 −/− (KO) mice with 762 (anti-SAM, D) and 376 (anti-HPS, E) antibodies. TANK1 indicates tankyrase 1 protein, TANK1x indicates a possible degraded tankyrase 1 protein, and TANK1a indicates tankyrase 1a protein.

    Article Snippet: RT-PCR reactions were carried out in 50 µl of PCR reaction mix containing 25 µl of PCR buffer (One-step RT-PCR kit, Life Technologies), 1 µg of total RNA and 0.2 µM primers.

    Techniques: Expressing, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Western Blot

    PGRN deficiency increased the production of systemic and local pro‐inflammatory mediators after injection of LPS . ELISA of serum levels of inflammatory mediators including tumour necrosis factor α ( TNF ‐α) ( A ), interleukin 6 ( IL ‐6) ( B ), IL ‐10 ( C ) and monocyte chemoattractant protein 1 ( MCP ‐1) ( D ). ( E ) Western blot analysis of cold‐inducible RNA ‐binding protein ( CIRP ) and high mobility group box protein 1 ( HMGB 1) in the serum from WT and Grn −/− mice with LPS injection at 6 and 24 hrs. PS red, Ponceau S red staining. Real‐time RT ‐ PCR analysis of mRNA levels of TNF ‐α ( F ), IL ‐6 ( G ), IL ‐1β ( H ) and IL ‐10 ( I ) in the lung after LPS injection. Data are mean ± S.D. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Progranulin deficiency leads to severe inflammation, lung injury and cell death in a mouse model of endotoxic shock

    doi: 10.1111/jcmm.12756

    Figure Lengend Snippet: PGRN deficiency increased the production of systemic and local pro‐inflammatory mediators after injection of LPS . ELISA of serum levels of inflammatory mediators including tumour necrosis factor α ( TNF ‐α) ( A ), interleukin 6 ( IL ‐6) ( B ), IL ‐10 ( C ) and monocyte chemoattractant protein 1 ( MCP ‐1) ( D ). ( E ) Western blot analysis of cold‐inducible RNA ‐binding protein ( CIRP ) and high mobility group box protein 1 ( HMGB 1) in the serum from WT and Grn −/− mice with LPS injection at 6 and 24 hrs. PS red, Ponceau S red staining. Real‐time RT ‐ PCR analysis of mRNA levels of TNF ‐α ( F ), IL ‐6 ( G ), IL ‐1β ( H ) and IL ‐10 ( I ) in the lung after LPS injection. Data are mean ± S.D. * P

    Article Snippet: Then, 1 μg of DNA‐free total RNA was reverse transcribed by use of a one‐step RT‐PCR kit (TaKaRa Bio, Shiga, Japan).

    Techniques: Injection, Enzyme-linked Immunosorbent Assay, Western Blot, RNA Binding Assay, Mouse Assay, Staining, Quantitative RT-PCR

    RT-PCR analysis of C. perfringens strains. (A) Total RNA prepared from each specified strain was subjected to RT-PCR analysis using cpb2 -specific internal primers as described in Materials and Methods. RT (+) and RT (−) indicate the presence

    Journal: Journal of Clinical Microbiology

    Article Title: Regulated Expression of the Beta2-Toxin Gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases

    doi: 10.1128/JCM.43.8.4002-4009.2005

    Figure Lengend Snippet: RT-PCR analysis of C. perfringens strains. (A) Total RNA prepared from each specified strain was subjected to RT-PCR analysis using cpb2 -specific internal primers as described in Materials and Methods. RT (+) and RT (−) indicate the presence

    Article Snippet: The primer set 5′-AAACTGAATTTTTAAATGGTGC-3′ and 5′-TCCACATCCAATGATCTACAA-3′, which amplified a 318-bp cpb2 internal fragment, was used to detect cpb2 -specific mRNA in total RNA preparations by reverse transcriptase (RT)-PCR analysis with the commercially available Access RT-PCR kit (Promega).

    Techniques: Reverse Transcription Polymerase Chain Reaction

    Effect of XWL-1-48 on PI3K/Akt/Mdm2 pathway. ( A ) HepG2 cells were treated by XWL-1-48 (0.1, 0.3, 1 μM) for 24 h, protein expression of p-Akt, Akt, p-Mdm2 and Mdm2 were determined by Western bolt analysis. The changes of p-Akt/Akt and p-ATM/ATM were quantified using Image J software. A representative result is shown from at least three independent experiments. ( B ) HepG2 cells were exposure to 1 μM of XWL-1-48 for 0, 2, 4, 6 12 and 24 h, protein expression level of Mdm2 was determined using immunoblot, and further quantified by image J software. ( C ) After treated with XWL-1-48 (0.1, 0.3, 1 μM) for 24 h, Cells were harvested. The mRNA level of Mdm2 was analyzed by RT-PCR. ( D ) After incubated with XWL-1-48 for 24 h, the expression of Mdm2 in cytoplasm and nuclear were detected. ( E ) HepG2 cells were exposed to 100 μg/ml cycloheximide (CHX, known as protein synthesis inhibitor) with or without XWL-1-48 (1 μM) for 2, 4, 6 h to block protein synthesis. The cells were collected for Western blot analysis. ( F ) Cells were treated for 48 h with or without XWL-1-48 (1 μM). Cell lysates were immunoprecipitated with 1 μg of anti-Mdm2 polyclonal antibody, followed by Western blotting with a monoclonal anti-ubiquitin antibody. A representative result is shown from at least three independent experiments. * p

    Journal: Scientific Reports

    Article Title: XWL-1-48 exerts antitumor activity via targeting topoisomerase II and enhancing degradation of Mdm2 in human hepatocellular carcinoma

    doi: 10.1038/s41598-017-10577-7

    Figure Lengend Snippet: Effect of XWL-1-48 on PI3K/Akt/Mdm2 pathway. ( A ) HepG2 cells were treated by XWL-1-48 (0.1, 0.3, 1 μM) for 24 h, protein expression of p-Akt, Akt, p-Mdm2 and Mdm2 were determined by Western bolt analysis. The changes of p-Akt/Akt and p-ATM/ATM were quantified using Image J software. A representative result is shown from at least three independent experiments. ( B ) HepG2 cells were exposure to 1 μM of XWL-1-48 for 0, 2, 4, 6 12 and 24 h, protein expression level of Mdm2 was determined using immunoblot, and further quantified by image J software. ( C ) After treated with XWL-1-48 (0.1, 0.3, 1 μM) for 24 h, Cells were harvested. The mRNA level of Mdm2 was analyzed by RT-PCR. ( D ) After incubated with XWL-1-48 for 24 h, the expression of Mdm2 in cytoplasm and nuclear were detected. ( E ) HepG2 cells were exposed to 100 μg/ml cycloheximide (CHX, known as protein synthesis inhibitor) with or without XWL-1-48 (1 μM) for 2, 4, 6 h to block protein synthesis. The cells were collected for Western blot analysis. ( F ) Cells were treated for 48 h with or without XWL-1-48 (1 μM). Cell lysates were immunoprecipitated with 1 μg of anti-Mdm2 polyclonal antibody, followed by Western blotting with a monoclonal anti-ubiquitin antibody. A representative result is shown from at least three independent experiments. * p

    Article Snippet: The integrity and purity of the RNA were evaluated by UV Spectrophotometer for OD260 and OD280, then reverse transcripted from mRNA to cDNA using the RT-PCR kit (Promega, WI, USA).

    Techniques: Expressing, Western Blot, Software, Reverse Transcription Polymerase Chain Reaction, Incubation, Blocking Assay, Immunoprecipitation