reverse transcriptase pcr qrt pcr kit  (Thermo Fisher)


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    Name:
    SuperScript III First Strand Synthesis SuperMix for qRT PCR
    Description:
    SuperScript III First Strand Synthesis SuperMix for qRT PCR provides the high temperature capability of SuperScript III Reverse Transcriptase in an optimized SuperMix format for the synthesis of first strand cDNA for use in real time quantitative RT PCR qRT PCR The simple time saving reaction set up uses just two tubes a 2X Reaction Mix and an Enzyme Mix Enzyme mixSuperScript III Reverse Transcriptase included in the RT Enzyme Mix is a version of M MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability The enzyme can be used to synthesize cDNA at a temperature range of 42 60°C providing increased specificity higher yields of cDNA and more full length product than other reverse transcriptases Because SuperScript III RT is not significantly inhibited by ribosomal and transfer RNA it can be used to synthesize cDNA from total RNA RNaseOUT Recombinant Ribonuclease Inhibitor also included in the enzyme mix is an RNase inhibitor protein that safeguards against the degradation of target RNA due to ribonuclease contamination of the RNA preparation Reaction mixThe 2X RT Reaction Mix includes oligo dT 20 random hexamers MgCl2 and dNTPs in a buffer formulation that has been optimized for qRT PCR E coli RNase H is provided as a separate tube in the kit to remove the RNA template from the cDNA RNA hybrid molecule after first strand synthesis This has been shown to increase sensitivity in qRT PCR Using SuperScript III First Strand Synthesis SuperMixThis SuperMix formulation can be used to quantify fewer than 10 copies of a target gene in qRT PCR with a broad dynamic range that supports accurate quantification of high copy mRNA from up to 1 µg of total RNA Reagents are provided for 50 or 250 RT reactions of 20 µL each
    Catalog Number:
    11752050
    Price:
    None
    Category:
    Kits and Assays
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
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    Structured Review

    Thermo Fisher reverse transcriptase pcr qrt pcr kit
    Microbiota independent inflammatory and stress response phenotypes of EPI-/- skin. ( a ) qPCR of 16s RNA gene in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- ear skin samples. ( b ) Number of CD4 + CD3 + cells per mm 2 dermis. ( c ) Number of γδTCR + cells per mm epidermis. ( d ) Number of Vγ3 + cells per mm epidermis. ( e ) <t>qRT-PCR</t> of Rae-1 in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- epidermis. ( f ) Quantification of MPO in WT, EPI-/-, and flora-deficient skin lysates. Data are means ± SEM from at least four ( a ) or three ( b–f ) mice per group. * P
    SuperScript III First Strand Synthesis SuperMix for qRT PCR provides the high temperature capability of SuperScript III Reverse Transcriptase in an optimized SuperMix format for the synthesis of first strand cDNA for use in real time quantitative RT PCR qRT PCR The simple time saving reaction set up uses just two tubes a 2X Reaction Mix and an Enzyme Mix Enzyme mixSuperScript III Reverse Transcriptase included in the RT Enzyme Mix is a version of M MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability The enzyme can be used to synthesize cDNA at a temperature range of 42 60°C providing increased specificity higher yields of cDNA and more full length product than other reverse transcriptases Because SuperScript III RT is not significantly inhibited by ribosomal and transfer RNA it can be used to synthesize cDNA from total RNA RNaseOUT Recombinant Ribonuclease Inhibitor also included in the enzyme mix is an RNase inhibitor protein that safeguards against the degradation of target RNA due to ribonuclease contamination of the RNA preparation Reaction mixThe 2X RT Reaction Mix includes oligo dT 20 random hexamers MgCl2 and dNTPs in a buffer formulation that has been optimized for qRT PCR E coli RNase H is provided as a separate tube in the kit to remove the RNA template from the cDNA RNA hybrid molecule after first strand synthesis This has been shown to increase sensitivity in qRT PCR Using SuperScript III First Strand Synthesis SuperMixThis SuperMix formulation can be used to quantify fewer than 10 copies of a target gene in qRT PCR with a broad dynamic range that supports accurate quantification of high copy mRNA from up to 1 µg of total RNA Reagents are provided for 50 or 250 RT reactions of 20 µL each
    https://www.bioz.com/result/reverse transcriptase pcr qrt pcr kit/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    reverse transcriptase pcr qrt pcr kit - by Bioz Stars, 2021-04
    99/100 stars

    Images

    1) Product Images from "Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility"

    Article Title: Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility

    Journal: The Journal of Investigative Dermatology

    doi: 10.1038/JID.2015.383

    Microbiota independent inflammatory and stress response phenotypes of EPI-/- skin. ( a ) qPCR of 16s RNA gene in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- ear skin samples. ( b ) Number of CD4 + CD3 + cells per mm 2 dermis. ( c ) Number of γδTCR + cells per mm epidermis. ( d ) Number of Vγ3 + cells per mm epidermis. ( e ) qRT-PCR of Rae-1 in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- epidermis. ( f ) Quantification of MPO in WT, EPI-/-, and flora-deficient skin lysates. Data are means ± SEM from at least four ( a ) or three ( b–f ) mice per group. * P
    Figure Legend Snippet: Microbiota independent inflammatory and stress response phenotypes of EPI-/- skin. ( a ) qPCR of 16s RNA gene in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- ear skin samples. ( b ) Number of CD4 + CD3 + cells per mm 2 dermis. ( c ) Number of γδTCR + cells per mm epidermis. ( d ) Number of Vγ3 + cells per mm epidermis. ( e ) qRT-PCR of Rae-1 in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- epidermis. ( f ) Quantification of MPO in WT, EPI-/-, and flora-deficient skin lysates. Data are means ± SEM from at least four ( a ) or three ( b–f ) mice per group. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mouse Assay

    AMP expression and protease activity in EPI-/- epidermis after microbiota modulation. ( a–k ) qRT-PCR of Camp ( a ) , Defb1 ( b ), Defb2 ( c ), Defb3 ( d ), Defb6 ( e ), S100A9 ( f ), Pla2g2a ( g ), Reg3b ( h ), Reg3g ( i ), and Serpina1b ( j ) in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- epidermis. Data are means ± SEM from at least four mice per group. * P
    Figure Legend Snippet: AMP expression and protease activity in EPI-/- epidermis after microbiota modulation. ( a–k ) qRT-PCR of Camp ( a ) , Defb1 ( b ), Defb2 ( c ), Defb3 ( d ), Defb6 ( e ), S100A9 ( f ), Pla2g2a ( g ), Reg3b ( h ), Reg3g ( i ), and Serpina1b ( j ) in WT, EPI-/-, antibiotic-treated EPI-/-, and flora-deficient EPI-/- epidermis. Data are means ± SEM from at least four mice per group. * P

    Techniques Used: Expressing, Activity Assay, Quantitative RT-PCR, Mouse Assay

    Related Articles

    Quantitative RT-PCR:

    Article Title: Genome-wide functional analysis of phosphatases in the pathogenic fungus Cryptococcus neoformans
    Article Snippet: Total RNA was extracted from each sample using a commercial RNA extraction kit (easy-BLUE, iNtRON Biotechnology, Gyeonggi, Korea) and cDNA was synthesized using RTase (Thermo Scientific, Waltham, MA, USA). .. Quantitative reverse transcription-PCR (qRT-PCR) was performed with target gene-specific primer pairs listed in Supplementary Data . .. HPLC analysis of O -linked glycans from cwMPs Analysis of O -linked glycans from cwMPs was conducted as described previously , .

    Article Title: Adenosine monophosphate-activated protein kinase (AMPK) restricts Zika virus replication in endothelial cells by potentiating innate antiviral responses and inhibiting glycolysis
    Article Snippet: .. RNA isolation and quantitative reverse transcriptase PCR (qRT-PCR). .. Total RNA was extracted from the cells infected and treated with the drugs as specified using Trizol (Invitrogen) reagent as per the manufacturer’s instructions.

    Article Title: Alteration of Fermentative Metabolism Enhances Mucor circinelloides Virulence
    Article Snippet: RNA samples were separated on nondenaturing 2% agarose gels stained with ethidium bromide, visualized using a Gel Doc XR+ imager (Bio-Rad, Hercules, CA, USA), and quantified using a SmartSpec Plus spectrophotometer (Bio-Rad). .. Oligonucleotide design and real-time quantitative reverse transcription-PCR (qRT-PCR). .. Primers and hydrolysis probes for the ald2 gene from M. circinelloides encoding the homolog to ALD2 from Saccharomyces cerevisiae were used as reported previously , and the β-actin ( Actβ ), interleukin 1β ( Il-1β ), interleukin 6 ( Il-6 ), and murine macrophage inflammatory protein 2 ( Mip2) genes from mice were used as described before ( ). qRT-PCR was performed using the LightCycler 480 II system (Roche Molecular Diagnostics, Pleasanton, CA, USA) employing the SuperScript III Platinum one-step qRT-PCR reagent kit (Invitrogen, Carlsbad, CA, USA).

    Article Title: Transcriptional regulation of homeostatic and disease-associated-microglial genes by IRF1, LXRβ and CEBPα
    Article Snippet: Primary microglia were transfected with 40nM (final concentration) of siRNA using Lipofectamine™ RNAiMAX (Invitrogen) and Opti-MEM (Invitrogen, Cat #31985–062). .. Cells were cultured for 48h before to confirm the efficiency of siRNA-mediated gene silencing by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). .. To activate microglia, lipopolysaccharide (LPS, final concentration 2ng/ml, #L4391, E. coli 0111:B4, Sigma-Aldrich) or recombinant mouse interferon γ (IFNγ #485-MI R & D Systems, final concentration 1–100ng/mL) was added after 24h of siRNA exposure and cells were collected after an additional 24h of activation for qRT-PCR, Nanostring and phagocytosis studies while supernatants were collected for cytokine assays.

    Article Title: PdtaS Deficiency Affects Resistance of Mycobacteria to Ribosome Targeting Antibiotics
    Article Snippet: The reverse transcription reaction and qRT-PCR analysis were performed as described previously by . .. Briefly, 1 μg of total RNA was reverse transcribed using specific primers to each studied gene (Supplementary Table ) and the SuperScript III First-Strand Synthesis SuperMix (Invitrogen). qRT-PCR analysis of expression levels of 16S, 23S and 5S genes was performed using the Maxima SYBR green qPCR master mix (Thermo Fisher Scientific). .. Each reaction mixture at a final volume of 25 μl contained 1 x Maxima SYBR green qPCR master mix, 50 ng of cDNA, and 0.3 M of each primer (see Supplementary Table for primer sequences).

    Article Title: Viral Determinants in H5N1 Influenza A Virus Enable Productive Infection of HeLa Cells
    Article Snippet: At 48 h posttransfection, either cells were infected, and productive virus growth was measured by plaque assay, or cell viability was determined using the CellTiter-Glo assay (Promega). .. RNA extraction and quantitative reverse transcription-PCR (qRT-PCR). .. At 48 h post-siRNA transfection, total RNA was extracted from HeLa cells using an RNeasy minikit (Qiagen).

    Isolation:

    Article Title: Adenosine monophosphate-activated protein kinase (AMPK) restricts Zika virus replication in endothelial cells by potentiating innate antiviral responses and inhibiting glycolysis
    Article Snippet: .. RNA isolation and quantitative reverse transcriptase PCR (qRT-PCR). .. Total RNA was extracted from the cells infected and treated with the drugs as specified using Trizol (Invitrogen) reagent as per the manufacturer’s instructions.

    Polymerase Chain Reaction:

    Article Title: Adenosine monophosphate-activated protein kinase (AMPK) restricts Zika virus replication in endothelial cells by potentiating innate antiviral responses and inhibiting glycolysis
    Article Snippet: .. RNA isolation and quantitative reverse transcriptase PCR (qRT-PCR). .. Total RNA was extracted from the cells infected and treated with the drugs as specified using Trizol (Invitrogen) reagent as per the manufacturer’s instructions.

    Article Title: Transcriptional regulation of homeostatic and disease-associated-microglial genes by IRF1, LXRβ and CEBPα
    Article Snippet: Primary microglia were transfected with 40nM (final concentration) of siRNA using Lipofectamine™ RNAiMAX (Invitrogen) and Opti-MEM (Invitrogen, Cat #31985–062). .. Cells were cultured for 48h before to confirm the efficiency of siRNA-mediated gene silencing by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). .. To activate microglia, lipopolysaccharide (LPS, final concentration 2ng/ml, #L4391, E. coli 0111:B4, Sigma-Aldrich) or recombinant mouse interferon γ (IFNγ #485-MI R & D Systems, final concentration 1–100ng/mL) was added after 24h of siRNA exposure and cells were collected after an additional 24h of activation for qRT-PCR, Nanostring and phagocytosis studies while supernatants were collected for cytokine assays.

    Cell Culture:

    Article Title: Transcriptional regulation of homeostatic and disease-associated-microglial genes by IRF1, LXRβ and CEBPα
    Article Snippet: Primary microglia were transfected with 40nM (final concentration) of siRNA using Lipofectamine™ RNAiMAX (Invitrogen) and Opti-MEM (Invitrogen, Cat #31985–062). .. Cells were cultured for 48h before to confirm the efficiency of siRNA-mediated gene silencing by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). .. To activate microglia, lipopolysaccharide (LPS, final concentration 2ng/ml, #L4391, E. coli 0111:B4, Sigma-Aldrich) or recombinant mouse interferon γ (IFNγ #485-MI R & D Systems, final concentration 1–100ng/mL) was added after 24h of siRNA exposure and cells were collected after an additional 24h of activation for qRT-PCR, Nanostring and phagocytosis studies while supernatants were collected for cytokine assays.

    Expressing:

    Article Title: PdtaS Deficiency Affects Resistance of Mycobacteria to Ribosome Targeting Antibiotics
    Article Snippet: The reverse transcription reaction and qRT-PCR analysis were performed as described previously by . .. Briefly, 1 μg of total RNA was reverse transcribed using specific primers to each studied gene (Supplementary Table ) and the SuperScript III First-Strand Synthesis SuperMix (Invitrogen). qRT-PCR analysis of expression levels of 16S, 23S and 5S genes was performed using the Maxima SYBR green qPCR master mix (Thermo Fisher Scientific). .. Each reaction mixture at a final volume of 25 μl contained 1 x Maxima SYBR green qPCR master mix, 50 ng of cDNA, and 0.3 M of each primer (see Supplementary Table for primer sequences).

    SYBR Green Assay:

    Article Title: PdtaS Deficiency Affects Resistance of Mycobacteria to Ribosome Targeting Antibiotics
    Article Snippet: The reverse transcription reaction and qRT-PCR analysis were performed as described previously by . .. Briefly, 1 μg of total RNA was reverse transcribed using specific primers to each studied gene (Supplementary Table ) and the SuperScript III First-Strand Synthesis SuperMix (Invitrogen). qRT-PCR analysis of expression levels of 16S, 23S and 5S genes was performed using the Maxima SYBR green qPCR master mix (Thermo Fisher Scientific). .. Each reaction mixture at a final volume of 25 μl contained 1 x Maxima SYBR green qPCR master mix, 50 ng of cDNA, and 0.3 M of each primer (see Supplementary Table for primer sequences).

    Real-time Polymerase Chain Reaction:

    Article Title: PdtaS Deficiency Affects Resistance of Mycobacteria to Ribosome Targeting Antibiotics
    Article Snippet: The reverse transcription reaction and qRT-PCR analysis were performed as described previously by . .. Briefly, 1 μg of total RNA was reverse transcribed using specific primers to each studied gene (Supplementary Table ) and the SuperScript III First-Strand Synthesis SuperMix (Invitrogen). qRT-PCR analysis of expression levels of 16S, 23S and 5S genes was performed using the Maxima SYBR green qPCR master mix (Thermo Fisher Scientific). .. Each reaction mixture at a final volume of 25 μl contained 1 x Maxima SYBR green qPCR master mix, 50 ng of cDNA, and 0.3 M of each primer (see Supplementary Table for primer sequences).

    RNA Extraction:

    Article Title: Viral Determinants in H5N1 Influenza A Virus Enable Productive Infection of HeLa Cells
    Article Snippet: At 48 h posttransfection, either cells were infected, and productive virus growth was measured by plaque assay, or cell viability was determined using the CellTiter-Glo assay (Promega). .. RNA extraction and quantitative reverse transcription-PCR (qRT-PCR). .. At 48 h post-siRNA transfection, total RNA was extracted from HeLa cells using an RNeasy minikit (Qiagen).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Proteomic identification of the proteins related to cigarette smoke-induced cardiac hypertrophy in spontaneously hypertensive rats
    Article Snippet: The concentration of total RNA was quantified by spectrophotometry (ND-1000; NanoDrop Technologies, Wilmington, DE). .. RNA was reverse transcribed to single-strand cDNA using SuperScript III First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific, Waltham, MA). .. The cDNA was subjected to quantitative PCR analysis with FastStart Universal Probe Master Mix (Roche, Basel, Switzerland) using primers specific for mRNAs encoding atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), collagen I, and collagen III genes, with an ABI 7300 Real-Time PCR system (Thermo Fisher Scientific), as described previously .

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    Thermo Fisher high capacity cdna reverse transcriptase kit
    Analysis of mRNA expression levels of galectins in normal and infected corneas by qRT-PCR. Complementary <t>DNA</t> was synthesized from 100 ng each of total RNA preparations of normal and infected corneas using the High-Capacity <t>cDNA</t> Reverse Transcriptase Kit, and PCR amplification was performed in triplicate using gene-specific primers for β-actin, Gal-1, -3, -7, -8, and -9 and a Taqman master mix according to the manufacturer's instructions. A threshold cycle value (C t ) was calculated from each amplification plot. Quantification data of each gene were normalized to the expression of β-actin, a value of 1.0 was given to the expression of each gene in the control cornea and the expression values for galectins in infected corneas were calculated as a change in expression level with respect to the control cornea. At least four corneas were pooled and considered one biological replica. N = 4 for all galectins. Data are plotted as mean ± SEM and analyzed using one-way ANOVA. * P
    High Capacity Cdna Reverse Transcriptase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii reverse transcriptase kit
    Identification and validation of gigaxonin as a miR-501 target decreasing MYH3 levels in primary muscle cells. Primary myoblasts were transfected with control antagomir or antagomir-501 and harvested after 48 h. RNA was extracted and used for <t>cDNA</t> synthesis and RNA-seq after DNase-treatment ( n =3). (A) Venn diagram showing the overlap between predicted target genes for miR-501 in mouse and human, based on TargetScan v6.2. The 11 transcripts that were significantly upregulated, predicted as miR-501 targets in mouse and human, and conserved among mammals were considered for further analysis. (B) qRT-PCR confirmation of six out of the 11 selected genes as potential miR-501 targets based on inhibition or overexpression of miR-501 in primary myoblasts, respectively. Cells were harvested 48 h after transfection with the antagomirs or miRNA mimics. Values are shown relative to transfections with control mimic or antagomir as indicated by the dashed line. n =11-12. (C) Primary myoblasts were transfected with pcDNA3.1 vector encoding N-terminally FLAG-tagged gigaxonin or empty vector, and differentiation was induced by serum withdrawal for 2 days. Densitometry shows MYH3 protein normalized to GAPDH or desmin. n =6. (D) Effect of the proteasome inhibitor MG-132 on MYH3 levels after gigaxonin overexpression. MG-132 was added to the media at the indicated time points and concentrations before harvesting. (E) The human 3′ UTR of GAN was cloned into the pmirGLO vector with (mut) or without (wt) a mutation of <t>three</t> nucleotides in the miR-501-binding site. Constructs were transfected into HEK293 cells and luciferase activity was measured after 48 h. Firefly luciferase activity was normalized to Renilla luciferase activity. n =5. (F) qRT-PCR analysis of Gan expression in FACS-sorted MPs or regenerating muscle (CTX TA) 4 days after CTX injection. RNA derived from the same experiment shown in Fig. 5 A. Data are presented as mean±s.e.m. All qRT-PCR data are normalized to 18S rRNA. * P
    Superscript Iii Reverse Transcriptase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 97 stars, based on 1 article reviews
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    99
    Thermo Fisher quantitative reverse transcriptase pcr qrt pcr total rna
    EphA6 expression is positively correlated with vascular invasion and neural invasion of CaP A. The expression of EphA6 mRNA in a cohort of 112 CaP tumor tissue samples and 58 benign prostate tissue samples from BPH patients was assessed by <t>qRT-PCR.</t> B-H. Correlation of EphA6 expression with clinical and histological parameters in prostate cancer patients: PSA (B), TNM (C), vascular invasion (D), neural invasion (E), age (F), Gleason score (G), and prostate volume (H)
    Quantitative Reverse Transcriptase Pcr Qrt Pcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative reverse transcriptase pcr qrt pcr total rna/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
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    Image Search Results


    Analysis of mRNA expression levels of galectins in normal and infected corneas by qRT-PCR. Complementary DNA was synthesized from 100 ng each of total RNA preparations of normal and infected corneas using the High-Capacity cDNA Reverse Transcriptase Kit, and PCR amplification was performed in triplicate using gene-specific primers for β-actin, Gal-1, -3, -7, -8, and -9 and a Taqman master mix according to the manufacturer's instructions. A threshold cycle value (C t ) was calculated from each amplification plot. Quantification data of each gene were normalized to the expression of β-actin, a value of 1.0 was given to the expression of each gene in the control cornea and the expression values for galectins in infected corneas were calculated as a change in expression level with respect to the control cornea. At least four corneas were pooled and considered one biological replica. N = 4 for all galectins. Data are plotted as mean ± SEM and analyzed using one-way ANOVA. * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Fingerprinting of Galectins in Normal, P. aeruginosa–Infected, and Chemically Burned Mouse Corneas

    doi: 10.1167/iovs.14-15338

    Figure Lengend Snippet: Analysis of mRNA expression levels of galectins in normal and infected corneas by qRT-PCR. Complementary DNA was synthesized from 100 ng each of total RNA preparations of normal and infected corneas using the High-Capacity cDNA Reverse Transcriptase Kit, and PCR amplification was performed in triplicate using gene-specific primers for β-actin, Gal-1, -3, -7, -8, and -9 and a Taqman master mix according to the manufacturer's instructions. A threshold cycle value (C t ) was calculated from each amplification plot. Quantification data of each gene were normalized to the expression of β-actin, a value of 1.0 was given to the expression of each gene in the control cornea and the expression values for galectins in infected corneas were calculated as a change in expression level with respect to the control cornea. At least four corneas were pooled and considered one biological replica. N = 4 for all galectins. Data are plotted as mean ± SEM and analyzed using one-way ANOVA. * P

    Article Snippet: Complementary DNA was synthesized from 100 ng total RNA using the High-Capacity cDNA Reverse Transcriptase Kit (Invitrogen).

    Techniques: Expressing, Infection, Quantitative RT-PCR, Synthesized, Polymerase Chain Reaction, Amplification

    Identification and validation of gigaxonin as a miR-501 target decreasing MYH3 levels in primary muscle cells. Primary myoblasts were transfected with control antagomir or antagomir-501 and harvested after 48 h. RNA was extracted and used for cDNA synthesis and RNA-seq after DNase-treatment ( n =3). (A) Venn diagram showing the overlap between predicted target genes for miR-501 in mouse and human, based on TargetScan v6.2. The 11 transcripts that were significantly upregulated, predicted as miR-501 targets in mouse and human, and conserved among mammals were considered for further analysis. (B) qRT-PCR confirmation of six out of the 11 selected genes as potential miR-501 targets based on inhibition or overexpression of miR-501 in primary myoblasts, respectively. Cells were harvested 48 h after transfection with the antagomirs or miRNA mimics. Values are shown relative to transfections with control mimic or antagomir as indicated by the dashed line. n =11-12. (C) Primary myoblasts were transfected with pcDNA3.1 vector encoding N-terminally FLAG-tagged gigaxonin or empty vector, and differentiation was induced by serum withdrawal for 2 days. Densitometry shows MYH3 protein normalized to GAPDH or desmin. n =6. (D) Effect of the proteasome inhibitor MG-132 on MYH3 levels after gigaxonin overexpression. MG-132 was added to the media at the indicated time points and concentrations before harvesting. (E) The human 3′ UTR of GAN was cloned into the pmirGLO vector with (mut) or without (wt) a mutation of three nucleotides in the miR-501-binding site. Constructs were transfected into HEK293 cells and luciferase activity was measured after 48 h. Firefly luciferase activity was normalized to Renilla luciferase activity. n =5. (F) qRT-PCR analysis of Gan expression in FACS-sorted MPs or regenerating muscle (CTX TA) 4 days after CTX injection. RNA derived from the same experiment shown in Fig. 5 A. Data are presented as mean±s.e.m. All qRT-PCR data are normalized to 18S rRNA. * P

    Journal: Development (Cambridge, England)

    Article Title: MicroRNA deep sequencing in two adult stem cell populations identifies miR-501 as a novel regulator of myosin heavy chain during muscle regeneration

    doi: 10.1242/dev.136051

    Figure Lengend Snippet: Identification and validation of gigaxonin as a miR-501 target decreasing MYH3 levels in primary muscle cells. Primary myoblasts were transfected with control antagomir or antagomir-501 and harvested after 48 h. RNA was extracted and used for cDNA synthesis and RNA-seq after DNase-treatment ( n =3). (A) Venn diagram showing the overlap between predicted target genes for miR-501 in mouse and human, based on TargetScan v6.2. The 11 transcripts that were significantly upregulated, predicted as miR-501 targets in mouse and human, and conserved among mammals were considered for further analysis. (B) qRT-PCR confirmation of six out of the 11 selected genes as potential miR-501 targets based on inhibition or overexpression of miR-501 in primary myoblasts, respectively. Cells were harvested 48 h after transfection with the antagomirs or miRNA mimics. Values are shown relative to transfections with control mimic or antagomir as indicated by the dashed line. n =11-12. (C) Primary myoblasts were transfected with pcDNA3.1 vector encoding N-terminally FLAG-tagged gigaxonin or empty vector, and differentiation was induced by serum withdrawal for 2 days. Densitometry shows MYH3 protein normalized to GAPDH or desmin. n =6. (D) Effect of the proteasome inhibitor MG-132 on MYH3 levels after gigaxonin overexpression. MG-132 was added to the media at the indicated time points and concentrations before harvesting. (E) The human 3′ UTR of GAN was cloned into the pmirGLO vector with (mut) or without (wt) a mutation of three nucleotides in the miR-501-binding site. Constructs were transfected into HEK293 cells and luciferase activity was measured after 48 h. Firefly luciferase activity was normalized to Renilla luciferase activity. n =5. (F) qRT-PCR analysis of Gan expression in FACS-sorted MPs or regenerating muscle (CTX TA) 4 days after CTX injection. RNA derived from the same experiment shown in Fig. 5 A. Data are presented as mean±s.e.m. All qRT-PCR data are normalized to 18S rRNA. * P

    Article Snippet: First-strand cDNA was synthesized using SuperScript III Reverse Transcriptase Kit (Thermo Fisher Scientific) with random hexamers according to the manufacturer's instructions.

    Techniques: Transfection, RNA Sequencing Assay, Quantitative RT-PCR, Inhibition, Over Expression, Plasmid Preparation, Clone Assay, Mutagenesis, Binding Assay, Construct, Luciferase, Activity Assay, Expressing, FACS, Injection, Derivative Assay

    Enhanced BMP signaling in RBPJK deficient MSCs. (A) Real time RT-PCR analysis reveals that expression of RBPJK was significantly inhibited by 90% in shRBPJK lentivirus infected MSCs (shRBPJK) at day 2 when compared to shRNA-lentivirus control MSCs (Co). (B) Luciferase assays showed a significant increase of BMP responsive reporter activity in shRBPJK MSCs and this transcriptional up-regulation is further enhanced by BMP-2 treatment. Data are means ± s.d. of three independent experiments performed in duplicate and all the results were normalized to internal control (*, p

    Journal: PLoS ONE

    Article Title: Deletion of RBPJK in Mesenchymal Stem Cells Enhances Osteogenic Activity by Up-Regulation of BMP Signaling

    doi: 10.1371/journal.pone.0135971

    Figure Lengend Snippet: Enhanced BMP signaling in RBPJK deficient MSCs. (A) Real time RT-PCR analysis reveals that expression of RBPJK was significantly inhibited by 90% in shRBPJK lentivirus infected MSCs (shRBPJK) at day 2 when compared to shRNA-lentivirus control MSCs (Co). (B) Luciferase assays showed a significant increase of BMP responsive reporter activity in shRBPJK MSCs and this transcriptional up-regulation is further enhanced by BMP-2 treatment. Data are means ± s.d. of three independent experiments performed in duplicate and all the results were normalized to internal control (*, p

    Article Snippet: Real time RT-PCR cDNA was synthesized from 1 μg total RNA using the SuperScript III reverse transcriptase kit (Invitrogen) in a final volume of 20 μl.

    Techniques: Quantitative RT-PCR, Expressing, Infection, shRNA, Luciferase, Activity Assay

    Deletion of RBPJK enhances cell spontaneous differentiation in regular MSC culture. Mesenchymal stem cells were infected with Control shRNA(Co) or RBPJK shRNA (shRBPJK) lentivirus before being harvested for alkaline phosphatase (ALP) staining, western blot, RT-PCR analysis and BrdU ELISA. (A) Western Blot showed a significant decrease of RBPJK protein expression at day 3 and 14 following shRBPJK lentiviral infection. (B) Fold change in protein level of RBPJK in Western blots was determined by measuring band intensity with ImageJ. (C) Deletion of RBPJK resulted in increased ALP staining at days 3 and 14, compared to controls (Co). Scale bars, 100μm. (D) ALP gene expression was increased at days 3 and 14, with maximal increase at day 14 in RT-PCR analysis. (E) BrdU ELISA to monitor both MSCs proliferation at days 3 and 14 after lentiviral infection. Data are means ± s.d. of three independent experiments performed in duplicate and the control gene expression level at day 3 was set at 1. (*, p

    Journal: PLoS ONE

    Article Title: Deletion of RBPJK in Mesenchymal Stem Cells Enhances Osteogenic Activity by Up-Regulation of BMP Signaling

    doi: 10.1371/journal.pone.0135971

    Figure Lengend Snippet: Deletion of RBPJK enhances cell spontaneous differentiation in regular MSC culture. Mesenchymal stem cells were infected with Control shRNA(Co) or RBPJK shRNA (shRBPJK) lentivirus before being harvested for alkaline phosphatase (ALP) staining, western blot, RT-PCR analysis and BrdU ELISA. (A) Western Blot showed a significant decrease of RBPJK protein expression at day 3 and 14 following shRBPJK lentiviral infection. (B) Fold change in protein level of RBPJK in Western blots was determined by measuring band intensity with ImageJ. (C) Deletion of RBPJK resulted in increased ALP staining at days 3 and 14, compared to controls (Co). Scale bars, 100μm. (D) ALP gene expression was increased at days 3 and 14, with maximal increase at day 14 in RT-PCR analysis. (E) BrdU ELISA to monitor both MSCs proliferation at days 3 and 14 after lentiviral infection. Data are means ± s.d. of three independent experiments performed in duplicate and the control gene expression level at day 3 was set at 1. (*, p

    Article Snippet: Real time RT-PCR cDNA was synthesized from 1 μg total RNA using the SuperScript III reverse transcriptase kit (Invitrogen) in a final volume of 20 μl.

    Techniques: Infection, shRNA, ALP Assay, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing

    EphA6 expression is positively correlated with vascular invasion and neural invasion of CaP A. The expression of EphA6 mRNA in a cohort of 112 CaP tumor tissue samples and 58 benign prostate tissue samples from BPH patients was assessed by qRT-PCR. B-H. Correlation of EphA6 expression with clinical and histological parameters in prostate cancer patients: PSA (B), TNM (C), vascular invasion (D), neural invasion (E), age (F), Gleason score (G), and prostate volume (H)

    Journal: Oncotarget

    Article Title: EphA6 promotes angiogenesis and prostate cancer metastasis and is associated with human prostate cancer progression

    doi:

    Figure Lengend Snippet: EphA6 expression is positively correlated with vascular invasion and neural invasion of CaP A. The expression of EphA6 mRNA in a cohort of 112 CaP tumor tissue samples and 58 benign prostate tissue samples from BPH patients was assessed by qRT-PCR. B-H. Correlation of EphA6 expression with clinical and histological parameters in prostate cancer patients: PSA (B), TNM (C), vascular invasion (D), neural invasion (E), age (F), Gleason score (G), and prostate volume (H)

    Article Snippet: Quantitative reverse transcriptase PCR (qRT-PCR) Total RNA (1 μg) was isolated using Trizol reagent and subjected to reverse transcription reactions with random hexamer primers using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). qRT-PCR was performed on a 7900HT Sequence Detection system (Applied Biosystems) using FastStart Universal SYBR Green Master kit (Roche Diagnostics, Indianapolis, IN) and TaqMan® Universal Master Mix (Applied Biosystems) following the manufacturer's instructions.

    Techniques: Expressing, Quantitative RT-PCR

    EphA6 mRNA and protein expression is up-regulated in CaP lymph node metastatic cell lines and CaP tumor tissues A. The mRNA expression of all currently known members of human Eph receptor and ephrins was detected and quantitated by qRT-PCR in CaP cell lines LNCaP, PC-3, PC-3M and their derivative lymph node metastatic lines LNCaP/LN3 and PC-3M/LN4. Immortalized prostate epithelial cell line P69 was used as a control cell line. GAPDH was used as a house keeping gene control. The heat-map depicts the mRNA expression of Eph and ephrin family members. B. The expression of EphA6 mRNA was also shown in bar graphs for clearer presentation. * P

    Journal: Oncotarget

    Article Title: EphA6 promotes angiogenesis and prostate cancer metastasis and is associated with human prostate cancer progression

    doi:

    Figure Lengend Snippet: EphA6 mRNA and protein expression is up-regulated in CaP lymph node metastatic cell lines and CaP tumor tissues A. The mRNA expression of all currently known members of human Eph receptor and ephrins was detected and quantitated by qRT-PCR in CaP cell lines LNCaP, PC-3, PC-3M and their derivative lymph node metastatic lines LNCaP/LN3 and PC-3M/LN4. Immortalized prostate epithelial cell line P69 was used as a control cell line. GAPDH was used as a house keeping gene control. The heat-map depicts the mRNA expression of Eph and ephrin family members. B. The expression of EphA6 mRNA was also shown in bar graphs for clearer presentation. * P

    Article Snippet: Quantitative reverse transcriptase PCR (qRT-PCR) Total RNA (1 μg) was isolated using Trizol reagent and subjected to reverse transcription reactions with random hexamer primers using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). qRT-PCR was performed on a 7900HT Sequence Detection system (Applied Biosystems) using FastStart Universal SYBR Green Master kit (Roche Diagnostics, Indianapolis, IN) and TaqMan® Universal Master Mix (Applied Biosystems) following the manufacturer's instructions.

    Techniques: Expressing, Quantitative RT-PCR