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AVE0991 attenuates neuroinflammation in the brains of SAMP8 mice. Eight-month-old SAMP8 mice were injected intraperitoneally with vehicle or AVE0991 (1, 3 or 10 mg/kg/day) for 30 days. Afterwards, mice were sacrificed for analysis. ( A ) The mRNA levels of Il1b in the brains were investigated by <t>qRT-PCR.</t> ( B ) The mRNA levels of Il6 in the brains were investigated by qRT-PCR. ( C ) The mRNA levels of Tnf in the brains were investigated by qRT-PCR. ( D ) The protein levels of IL-1β in the brains were investigated by ELISA. ( E ) The protein levels of IL-6 in the brains were investigated by ELISA. ( F ) The protein levels of TNF-α in the brains were investigated by ELISA. In panel A - C, Gapdh was used as an internal control, and data were expressed as a fold change relative to non-treated SAMP8 mice. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=8 per group). # P
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1) Product Images from "AVE0991, a nonpeptide analogue of Ang-(1-7), attenuates aging-related neuroinflammation"

Article Title: AVE0991, a nonpeptide analogue of Ang-(1-7), attenuates aging-related neuroinflammation

Journal: Aging (Albany NY)

doi: 10.18632/aging/101419

AVE0991 attenuates neuroinflammation in the brains of SAMP8 mice. Eight-month-old SAMP8 mice were injected intraperitoneally with vehicle or AVE0991 (1, 3 or 10 mg/kg/day) for 30 days. Afterwards, mice were sacrificed for analysis. ( A ) The mRNA levels of Il1b in the brains were investigated by qRT-PCR. ( B ) The mRNA levels of Il6 in the brains were investigated by qRT-PCR. ( C ) The mRNA levels of Tnf in the brains were investigated by qRT-PCR. ( D ) The protein levels of IL-1β in the brains were investigated by ELISA. ( E ) The protein levels of IL-6 in the brains were investigated by ELISA. ( F ) The protein levels of TNF-α in the brains were investigated by ELISA. In panel A - C, Gapdh was used as an internal control, and data were expressed as a fold change relative to non-treated SAMP8 mice. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=8 per group). # P
Figure Legend Snippet: AVE0991 attenuates neuroinflammation in the brains of SAMP8 mice. Eight-month-old SAMP8 mice were injected intraperitoneally with vehicle or AVE0991 (1, 3 or 10 mg/kg/day) for 30 days. Afterwards, mice were sacrificed for analysis. ( A ) The mRNA levels of Il1b in the brains were investigated by qRT-PCR. ( B ) The mRNA levels of Il6 in the brains were investigated by qRT-PCR. ( C ) The mRNA levels of Tnf in the brains were investigated by qRT-PCR. ( D ) The protein levels of IL-1β in the brains were investigated by ELISA. ( E ) The protein levels of IL-6 in the brains were investigated by ELISA. ( F ) The protein levels of TNF-α in the brains were investigated by ELISA. In panel A - C, Gapdh was used as an internal control, and data were expressed as a fold change relative to non-treated SAMP8 mice. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=8 per group). # P

Techniques Used: Mouse Assay, Injection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Ang-(1-7) levels are decreased in the brains of SAMP8 mice during the aging process. ( A ) The activity of ACE2 in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were assessed using a specific detection kit. ( B ) The Ang-(1-7) levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were detected by ELISA. ( C ) The expression of Mas1 mRNA levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were assessed by qRT-PCR. Gapdh was used as an internal control. Data from panel B and C were expressed as a fold change relative to 4-month-old SAMR1 control mice. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=8 per group). * P
Figure Legend Snippet: Ang-(1-7) levels are decreased in the brains of SAMP8 mice during the aging process. ( A ) The activity of ACE2 in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were assessed using a specific detection kit. ( B ) The Ang-(1-7) levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were detected by ELISA. ( C ) The expression of Mas1 mRNA levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were assessed by qRT-PCR. Gapdh was used as an internal control. Data from panel B and C were expressed as a fold change relative to 4-month-old SAMR1 control mice. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=8 per group). * P

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

AVE0991 elevates microglial M2 activation makers in the brains of SAMP8 mice. Eight-month-old SAMP8 mice were injected intraperitoneally with vehicle or AVE0991 (1, 3 or 10 mg/kg/day) for 30 days. Afterwards, mice were sacrificed for analysis. ( A ) The mRNA levels of Arg1 in the brains were investigated by qRT-PCR. ( B ) The mRNA levels of Il10 in the brains were investigated by qRT-PCR. ( C ) The mRNA levels of Retnla in the brains were investigated by qRT-PCR. Gapdh was used as an internal control, and data were expressed as a fold change relative to non-treated SAMP8 mice. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=8 per group). # P
Figure Legend Snippet: AVE0991 elevates microglial M2 activation makers in the brains of SAMP8 mice. Eight-month-old SAMP8 mice were injected intraperitoneally with vehicle or AVE0991 (1, 3 or 10 mg/kg/day) for 30 days. Afterwards, mice were sacrificed for analysis. ( A ) The mRNA levels of Arg1 in the brains were investigated by qRT-PCR. ( B ) The mRNA levels of Il10 in the brains were investigated by qRT-PCR. ( C ) The mRNA levels of Retnla in the brains were investigated by qRT-PCR. Gapdh was used as an internal control, and data were expressed as a fold change relative to non-treated SAMP8 mice. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=8 per group). # P

Techniques Used: Activation Assay, Mouse Assay, Injection, Quantitative RT-PCR

AVE0991 suppresses microglial-mediated inflammatory response through a MAS1 receptor-dependent manner. Primary microglia were directly isolated from the brain of 8-month-old SAMP8 mice. ( A ) The localization of MAS1 receptor on microglia was confirmed by double immunofluorescence staining. Scale bar=15 μm. Next, microglia were treated for 4 h with 100 ng/ml LPS with or without 4 h pre-incubation with AVE0991 (1×10 -8 , 1×10 -7 or 1×10 -6 M) in the presence or absence of A-779 (1×10 -6 M) and were harvested and lysed for analysis. ( B ) The mRNA levels of Il6 were investigated by qRT-PCR. ( C ) The mRNA levels of Ilb were investigated by qRT-PCR. ( D ) The mRNA levels of Tnf were investigated by qRT-PCR. In panel B - D, Gapdh was used as an internal control, and data were expressed as a fold change relative to non-treated microglia. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=4-6). * P
Figure Legend Snippet: AVE0991 suppresses microglial-mediated inflammatory response through a MAS1 receptor-dependent manner. Primary microglia were directly isolated from the brain of 8-month-old SAMP8 mice. ( A ) The localization of MAS1 receptor on microglia was confirmed by double immunofluorescence staining. Scale bar=15 μm. Next, microglia were treated for 4 h with 100 ng/ml LPS with or without 4 h pre-incubation with AVE0991 (1×10 -8 , 1×10 -7 or 1×10 -6 M) in the presence or absence of A-779 (1×10 -6 M) and were harvested and lysed for analysis. ( B ) The mRNA levels of Il6 were investigated by qRT-PCR. ( C ) The mRNA levels of Ilb were investigated by qRT-PCR. ( D ) The mRNA levels of Tnf were investigated by qRT-PCR. In panel B - D, Gapdh was used as an internal control, and data were expressed as a fold change relative to non-treated microglia. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=4-6). * P

Techniques Used: Isolation, Mouse Assay, Double Immunofluorescence Staining, Incubation, Quantitative RT-PCR

AVE0991 elevates M2 activation makers in primary microglia. Primary microglia were directly isolated from the brain of 8-month-old SAMP8 mice. They were treated for 4 h with 100 ng/ml LPS with or without 4 h pre-incubation with AVE0991 (1×10 -8 , 1×10 -7 or 1×10 -6 M) in the presence or absence of A-779 (1×10 -6 M) and were harvested and lysed for analysis. ( A ) The mRNA levels of Arg1 were investigated by qRT-PCR. ( B ) The mRNA levels of Il10 were investigated by qRT-PCR. ( C ) The mRNA levels of Retnla were investigated by qRT-PCR. Gapdh was used as an internal control, and data were expressed as a fold change relative to non-treated microglia. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=4-6). * P
Figure Legend Snippet: AVE0991 elevates M2 activation makers in primary microglia. Primary microglia were directly isolated from the brain of 8-month-old SAMP8 mice. They were treated for 4 h with 100 ng/ml LPS with or without 4 h pre-incubation with AVE0991 (1×10 -8 , 1×10 -7 or 1×10 -6 M) in the presence or absence of A-779 (1×10 -6 M) and were harvested and lysed for analysis. ( A ) The mRNA levels of Arg1 were investigated by qRT-PCR. ( B ) The mRNA levels of Il10 were investigated by qRT-PCR. ( C ) The mRNA levels of Retnla were investigated by qRT-PCR. Gapdh was used as an internal control, and data were expressed as a fold change relative to non-treated microglia. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Columns represent mean ± SD (n=4-6). * P

Techniques Used: Activation Assay, Isolation, Mouse Assay, Incubation, Quantitative RT-PCR

Inflammatory markers are increased in the brains of SAMP8 mice during the aging process. ( A ) The dynamic changes of Il1b mRNA levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were investigated by qRT-PCR. ( B ) The dynamic changes of Il6 mRNA levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were investigated by qRT-PCR. ( C ) The dynamic changes of Tnf mRNA levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were investigated by qRT-PCR. Gapdh was used as an internal control. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test and were expressed as a fold change relative to 4-month-old SAMR1 control mice. Columns represent mean ± SD (n=8 per group). * P
Figure Legend Snippet: Inflammatory markers are increased in the brains of SAMP8 mice during the aging process. ( A ) The dynamic changes of Il1b mRNA levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were investigated by qRT-PCR. ( B ) The dynamic changes of Il6 mRNA levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were investigated by qRT-PCR. ( C ) The dynamic changes of Tnf mRNA levels in the brains of 4-, 8-, and 12-month-old SAMP8 mice as well as their age-matched SAMR1 control mice were investigated by qRT-PCR. Gapdh was used as an internal control. All data were analyzed by one-way ANOVA followed by Tukey’s post hoc test and were expressed as a fold change relative to 4-month-old SAMR1 control mice. Columns represent mean ± SD (n=8 per group). * P

Techniques Used: Mouse Assay, Quantitative RT-PCR

2) Product Images from "Heme acts through the Bach1b/Nrf2a-MafK pathway to regulate exocrine peptidase precursor genes in porphyric zebrafish"

Article Title: Heme acts through the Bach1b/Nrf2a-MafK pathway to regulate exocrine peptidase precursor genes in porphyric zebrafish

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.014951

Zebrafish bach1b is a co-ortholog of mammalian Bach1 , and heme regulates exocrine peptidase precursor genes. (A) Zebrafish Bach1b contains four CP motifs, three of which are also found in human and mouse. (B) Phylogenetic analysis of Bach1 proteins shows that zebrafish contain two bach1 genes, bach1a and bach1b , which are co-orthologs of mammalian Bach1 . The tree was constructed using the neighbor-joining method with MEGA5 ( Tamura et al., 2011 ). The numbers indicate the percentage bootstrap support. Dr , Danio rerio; Tr , Takifugu rubripes; Tn , Tetraodon nigroviridis; Ol , Oryzias latipes; Ga , Gasterosteus aculeatus; On , Oreochromis niloticus; Xt , Xenopus tropicalis; Ci , Ciona intestinalis; Cs , Ciona savignyi; Rn , Rattus norvegicus; Mm , Mus musculus; Hs , Homo sapiens. Ciona intestinalis and Ciona savignyi Bach proteins were used as an outgroup. The Ensembl gene IDs of these genes are listed in supplementary material Table S2 . (C) Downregulation of six peptidase precursor genes in zebrafish yquem/urod (−/−) and their rescue upon treatment with hemin. The concentration of hemin used was 100 μM. The expression levels of six peptidase precursor genes in larvae at 84 hpf were determined by using qRT-PCR. Student’s t -tests were conducted. * P
Figure Legend Snippet: Zebrafish bach1b is a co-ortholog of mammalian Bach1 , and heme regulates exocrine peptidase precursor genes. (A) Zebrafish Bach1b contains four CP motifs, three of which are also found in human and mouse. (B) Phylogenetic analysis of Bach1 proteins shows that zebrafish contain two bach1 genes, bach1a and bach1b , which are co-orthologs of mammalian Bach1 . The tree was constructed using the neighbor-joining method with MEGA5 ( Tamura et al., 2011 ). The numbers indicate the percentage bootstrap support. Dr , Danio rerio; Tr , Takifugu rubripes; Tn , Tetraodon nigroviridis; Ol , Oryzias latipes; Ga , Gasterosteus aculeatus; On , Oreochromis niloticus; Xt , Xenopus tropicalis; Ci , Ciona intestinalis; Cs , Ciona savignyi; Rn , Rattus norvegicus; Mm , Mus musculus; Hs , Homo sapiens. Ciona intestinalis and Ciona savignyi Bach proteins were used as an outgroup. The Ensembl gene IDs of these genes are listed in supplementary material Table S2 . (C) Downregulation of six peptidase precursor genes in zebrafish yquem/urod (−/−) and their rescue upon treatment with hemin. The concentration of hemin used was 100 μM. The expression levels of six peptidase precursor genes in larvae at 84 hpf were determined by using qRT-PCR. Student’s t -tests were conducted. * P

Techniques Used: Construct, Concentration Assay, Expressing, Quantitative RT-PCR

ChIP assays. Capped mRNAs encoding mafK -FLAG, nrf2a -FLAG or bach1b -HA were microinjected into one-cell stage embryos, antibodies against FLAG or HA were used to pull down complexes of the protein with the DNA, and the DNAs were eluted. Specific primers and the eluted DNAs were used in PCR analyses to amplify the DNA fragments that contained cpa5 MARE sites, which were subsequently quantified by using qRT-PCR. (A) Electrophoresis analysis of the ChIP results showed that MafK bound to MARE-containing regulatory fragments of six zymogens. (B) Electrophoresis analysis of the cpa5 ChIP assay. Approximately 2.6-fold more Nrf2a than Bach1b protein (quantified with ImageJ) was associated with cpa5 promoters that harbored MARE sites in wild-type control larvae; whereas approximately 2.8-fold more Bach1b than Nrf2a protein (quantified with ImageJ) was associated with cpa5 promoters that harbored MARE sites in heme-deficient yquem/urod (−/−) larvae. Moreover, treatment of the heme-deficient yquem/urod (−/−) larvae with hemin reversed the situation so that approximately 1.2-fold more Nrf2a than Bach1b protein (quantified with ImageJ) was associated with cpa5 promoters that harbored MARE sites in hemin-treated yquem/urod (−/−) larvae. (C) qRT-PCR analysis of the cpa5 ChIP assay, the results of which were consistent with the gel electrophoresis analysis in B.
Figure Legend Snippet: ChIP assays. Capped mRNAs encoding mafK -FLAG, nrf2a -FLAG or bach1b -HA were microinjected into one-cell stage embryos, antibodies against FLAG or HA were used to pull down complexes of the protein with the DNA, and the DNAs were eluted. Specific primers and the eluted DNAs were used in PCR analyses to amplify the DNA fragments that contained cpa5 MARE sites, which were subsequently quantified by using qRT-PCR. (A) Electrophoresis analysis of the ChIP results showed that MafK bound to MARE-containing regulatory fragments of six zymogens. (B) Electrophoresis analysis of the cpa5 ChIP assay. Approximately 2.6-fold more Nrf2a than Bach1b protein (quantified with ImageJ) was associated with cpa5 promoters that harbored MARE sites in wild-type control larvae; whereas approximately 2.8-fold more Bach1b than Nrf2a protein (quantified with ImageJ) was associated with cpa5 promoters that harbored MARE sites in heme-deficient yquem/urod (−/−) larvae. Moreover, treatment of the heme-deficient yquem/urod (−/−) larvae with hemin reversed the situation so that approximately 1.2-fold more Nrf2a than Bach1b protein (quantified with ImageJ) was associated with cpa5 promoters that harbored MARE sites in hemin-treated yquem/urod (−/−) larvae. (C) qRT-PCR analysis of the cpa5 ChIP assay, the results of which were consistent with the gel electrophoresis analysis in B.

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Quantitative RT-PCR, Electrophoresis, Nucleic Acid Electrophoresis

The regulatory functions of BACH1 on exocrine zymogens are conserved, developmental delay of the exocrine pancreas in the HEP fish and a model for heme-mediated regulation of the exocrine peptidase precursor gene. (A) Overexpression of human BACH1 in zebrafish resulted in downregulation of the six peptidase precursor genes investigated. (B) Downregulation of exdpf ( Jiang et al., 2008 ), an exocrine pancreas marker, in zebrafish yquem/urod (−/−), as determined by qRT-PCR analysis, the results of which are consistent with those achieved by using in situ hybridization (shown in supplementary material Fig. S7A,B ). The primers used for the qRT-PCR analysis are listed in supplementary material Table S1 . Student’s t -tests were conducted. ** P
Figure Legend Snippet: The regulatory functions of BACH1 on exocrine zymogens are conserved, developmental delay of the exocrine pancreas in the HEP fish and a model for heme-mediated regulation of the exocrine peptidase precursor gene. (A) Overexpression of human BACH1 in zebrafish resulted in downregulation of the six peptidase precursor genes investigated. (B) Downregulation of exdpf ( Jiang et al., 2008 ), an exocrine pancreas marker, in zebrafish yquem/urod (−/−), as determined by qRT-PCR analysis, the results of which are consistent with those achieved by using in situ hybridization (shown in supplementary material Fig. S7A,B ). The primers used for the qRT-PCR analysis are listed in supplementary material Table S1 . Student’s t -tests were conducted. ** P

Techniques Used: Fluorescence In Situ Hybridization, Over Expression, Marker, Quantitative RT-PCR, In Situ Hybridization

Knockdown experiments with bach1b and nrf2a reveal their antagonism in the regulation of zymogen expression. (A) qRT-PCR analysis showed that bach1b knockdown results in the upregulation of the six peptidase precursor genes that we examined, whereas nrf2a knockdown resulted in their downregulation. Knockdown experiments were performed by microinjecting MOs that targeted bach1b or nrf2a into one-cell stage embryos. For bach1b knockdown experiments, an ATG MO and SPL MO were used. Reverse transcription PCR showed that the SPL MO effectively altered bach1b intron 2 splicing ( supplementary material Fig. S1A,B ). The ATG MO also effectively knocked down bach1b ( supplementary material Fig. S1C ). The results using the SPL MO are shown here. For nrf2a knockdown experiments, we used an ATG MO, of which the efficacy of knockdown has been confirmed previously ( Kobayashi et al., 2002 ; Kobayashi et al., 2009 ; Wang and Gallagher, 2013 ). The expression levels of the six peptidase precursor genes in the microinjected and control larvae at 84 hpf were determined by using qRT-PCR. (B) Representative images of in situ hybridization staining show that the upregulation of cpa5 resulted from bach1b knockdown and that its downregulation resulted from nrf2a knockdown, both results occurred specifically in the exocrine pancreas. Dorsal view, anterior to the left. (C) Mean optic densities of in situ hybridization staining of a group of larvae (10–12 each) corresponding to Fig. 4C were quantified by using ImageJ. (D) cpa5 morphant phenotypes. For individual larvae (84 hpf) of the bach1b knockdown overexpression group, the optic density values higher than the mean optic density value of its own group were marked as ‘strong’, and the optic density values lower than the mean optic density of the control group were marked as ‘weak’. For individual larvae of the nrf2a knockdown group, the optic density values lower than the mean optic density value of its own group were marked as ‘strong’, the optic density values higher than the mean optic density of the control group were marked as ‘weak’, and the values of optic density between ‘weak’ and ‘strong’ were marked as ‘medium’. The statistical significance of difference between means was determined by using one-way ANOVA and Tukey’s multiple comparison test ( n =9) with SPSS10.0.1. * P
Figure Legend Snippet: Knockdown experiments with bach1b and nrf2a reveal their antagonism in the regulation of zymogen expression. (A) qRT-PCR analysis showed that bach1b knockdown results in the upregulation of the six peptidase precursor genes that we examined, whereas nrf2a knockdown resulted in their downregulation. Knockdown experiments were performed by microinjecting MOs that targeted bach1b or nrf2a into one-cell stage embryos. For bach1b knockdown experiments, an ATG MO and SPL MO were used. Reverse transcription PCR showed that the SPL MO effectively altered bach1b intron 2 splicing ( supplementary material Fig. S1A,B ). The ATG MO also effectively knocked down bach1b ( supplementary material Fig. S1C ). The results using the SPL MO are shown here. For nrf2a knockdown experiments, we used an ATG MO, of which the efficacy of knockdown has been confirmed previously ( Kobayashi et al., 2002 ; Kobayashi et al., 2009 ; Wang and Gallagher, 2013 ). The expression levels of the six peptidase precursor genes in the microinjected and control larvae at 84 hpf were determined by using qRT-PCR. (B) Representative images of in situ hybridization staining show that the upregulation of cpa5 resulted from bach1b knockdown and that its downregulation resulted from nrf2a knockdown, both results occurred specifically in the exocrine pancreas. Dorsal view, anterior to the left. (C) Mean optic densities of in situ hybridization staining of a group of larvae (10–12 each) corresponding to Fig. 4C were quantified by using ImageJ. (D) cpa5 morphant phenotypes. For individual larvae (84 hpf) of the bach1b knockdown overexpression group, the optic density values higher than the mean optic density value of its own group were marked as ‘strong’, and the optic density values lower than the mean optic density of the control group were marked as ‘weak’. For individual larvae of the nrf2a knockdown group, the optic density values lower than the mean optic density value of its own group were marked as ‘strong’, the optic density values higher than the mean optic density of the control group were marked as ‘weak’, and the values of optic density between ‘weak’ and ‘strong’ were marked as ‘medium’. The statistical significance of difference between means was determined by using one-way ANOVA and Tukey’s multiple comparison test ( n =9) with SPSS10.0.1. * P

Techniques Used: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, In Situ Hybridization, Staining, Over Expression

Overexpression experiments with bach1b and nrf2a reveal their antagonism in the regulation of zymogen expression. (A) qRT-PCR analysis showed that bach1b overexpression resulted in the downregulation of six peptidase precursor genes – cpa5 , ctr1l , ctrb1 , ela2l , try and tryl – whereas nrf2a overexpression resulted in their upregulation. Overexpression experiments were performed by microinjecting bach1b or nrf2a capped mRNAs into one-cell stage embryos. The expression levels of the six peptidase precursor genes in the microinjected and control larvae at 84 hpf were determined by qRT-PCR analysis. (B) Representative images of in situ hybridization staining show that downregulation of cpa5 resulted from bach1b overexpression and that its upregulation resulted from nrf2a overexpression, both specifically in the exocrine pancreas. Dorsal view, anterior to the left. (C) Mean optic densities of in situ hybridization staining of a group of larvae (10–12 each) corresponding to Fig. 3B were quantified by using ImageJ. (D) cpa5 morphant phenotypes. For individual larvae (84 hpf) of the bach1b overexpression group, the optic density values lower than the mean optic density value of its own group were marked as ‘strong’, and the optic density values higher than the mean optic density of the control group were marked as ‘weak’. For individual larvae of the nrf2a overexpression group, the optic density values higher than the mean optic density value of its own group were marked as ‘strong’, the optic density values lower than the mean optic density of the control group were marked as ‘weak’, and the values of optic density between ‘weak’ and ‘strong’ marked as ‘medium’. The statistical significance of difference between means was determined by one-way ANOVA and Tukey’s multiple comparison test ( n =9) by using SPSS10.0.1. * P
Figure Legend Snippet: Overexpression experiments with bach1b and nrf2a reveal their antagonism in the regulation of zymogen expression. (A) qRT-PCR analysis showed that bach1b overexpression resulted in the downregulation of six peptidase precursor genes – cpa5 , ctr1l , ctrb1 , ela2l , try and tryl – whereas nrf2a overexpression resulted in their upregulation. Overexpression experiments were performed by microinjecting bach1b or nrf2a capped mRNAs into one-cell stage embryos. The expression levels of the six peptidase precursor genes in the microinjected and control larvae at 84 hpf were determined by qRT-PCR analysis. (B) Representative images of in situ hybridization staining show that downregulation of cpa5 resulted from bach1b overexpression and that its upregulation resulted from nrf2a overexpression, both specifically in the exocrine pancreas. Dorsal view, anterior to the left. (C) Mean optic densities of in situ hybridization staining of a group of larvae (10–12 each) corresponding to Fig. 3B were quantified by using ImageJ. (D) cpa5 morphant phenotypes. For individual larvae (84 hpf) of the bach1b overexpression group, the optic density values lower than the mean optic density value of its own group were marked as ‘strong’, and the optic density values higher than the mean optic density of the control group were marked as ‘weak’. For individual larvae of the nrf2a overexpression group, the optic density values higher than the mean optic density value of its own group were marked as ‘strong’, the optic density values lower than the mean optic density of the control group were marked as ‘weak’, and the values of optic density between ‘weak’ and ‘strong’ marked as ‘medium’. The statistical significance of difference between means was determined by one-way ANOVA and Tukey’s multiple comparison test ( n =9) by using SPSS10.0.1. * P

Techniques Used: Over Expression, Expressing, Quantitative RT-PCR, In Situ Hybridization, Staining

3) Product Images from "PDL1 And LDHA act as ceRNAs in triple negative breast cancer by regulating miR-34a"

Article Title: PDL1 And LDHA act as ceRNAs in triple negative breast cancer by regulating miR-34a

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-017-0593-2

PDL1 is a target of miR-34a, and its functions could be inhibited by miR-34a. a The expression level of miR-34a was determined by qRT-PCR in the above cell lines. U6 snRNA was used as an internal control. b HCC38 and MDA-MB-231 cells were transfected with miR-34a mimic or scrambled oligonucleotide, and qRT-PCR analysis demonstrated that the transfection was successful. c HCC38 and MDA-MB-231 cells were transfected as described, and the mRNA and protein expression of PDL1 was suppressed by miR-34a. d Histogram presenting cell viability based on MTS assays for HCC38 and MDA-MB-231 cells 48 h after transfection. e Transwell invasion assays demonstrated that the PDL1 3’UTR promoted cell invasion. Representative images of invaded cells are shown in the left panel, and the results are summarized in the right panel. f The expression levels of PDL1 were determined by Western blotting in xenograft tumors (six in each group). β-Actin was used as an internal control. g The impact of miR-34a on immune cell populations in the tumor microenvironment. Flow cytometry revealed that miR-34a increased the number of CD8+ cells and CD4+ cells and reduced the number of macrophages and Tregs. All the data are shown as the mean ± s.e.m. * P
Figure Legend Snippet: PDL1 is a target of miR-34a, and its functions could be inhibited by miR-34a. a The expression level of miR-34a was determined by qRT-PCR in the above cell lines. U6 snRNA was used as an internal control. b HCC38 and MDA-MB-231 cells were transfected with miR-34a mimic or scrambled oligonucleotide, and qRT-PCR analysis demonstrated that the transfection was successful. c HCC38 and MDA-MB-231 cells were transfected as described, and the mRNA and protein expression of PDL1 was suppressed by miR-34a. d Histogram presenting cell viability based on MTS assays for HCC38 and MDA-MB-231 cells 48 h after transfection. e Transwell invasion assays demonstrated that the PDL1 3’UTR promoted cell invasion. Representative images of invaded cells are shown in the left panel, and the results are summarized in the right panel. f The expression levels of PDL1 were determined by Western blotting in xenograft tumors (six in each group). β-Actin was used as an internal control. g The impact of miR-34a on immune cell populations in the tumor microenvironment. Flow cytometry revealed that miR-34a increased the number of CD8+ cells and CD4+ cells and reduced the number of macrophages and Tregs. All the data are shown as the mean ± s.e.m. * P

Techniques Used: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification, Transfection, Western Blot, Flow Cytometry, Cytometry

LDHA is highly expressed in TNBC and correlated with a poor outcome. a The expression level of LDHA was determined by qRT-PCR and Western blotting in the above cell lines. β-Actin was used as an internal control. b The expression levels of LDHA in 20 pairs of TNBC tissues and their matched normal adjacent tissues. c OS (left) and DFS (right) curves for breast cancer patients with positive or negative LDHA expression. d OS (left) and DFS (right) curves for TNBC patients with positive or negative LDHA expression. All the data are shown as the mean ± s.e.m. * P
Figure Legend Snippet: LDHA is highly expressed in TNBC and correlated with a poor outcome. a The expression level of LDHA was determined by qRT-PCR and Western blotting in the above cell lines. β-Actin was used as an internal control. b The expression levels of LDHA in 20 pairs of TNBC tissues and their matched normal adjacent tissues. c OS (left) and DFS (right) curves for breast cancer patients with positive or negative LDHA expression. d OS (left) and DFS (right) curves for TNBC patients with positive or negative LDHA expression. All the data are shown as the mean ± s.e.m. * P

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

PDL1 is highly expressed in TNBC and correlated with a poor outcome. a The expression level of PDL1 was determined by qRT-PCR and Western blotting in seven different mammary cell lines, including one HME cell line (MCF-10A) and six TNBC cell lines. PDL1 expression was normalized using β-actin expression. b The expression level of PDL1 in 20 pairs of TNBC tissues and their matched normal adjacent tissues. c OS (left) and DFS (right) curves for breast cancer patients with positive or negative PDL1 expression. d OS (left) and DFS (right) curves for TNBC patients with positive or negative PDL1 expression. All the data are shown as the mean ± s.e.m. * P
Figure Legend Snippet: PDL1 is highly expressed in TNBC and correlated with a poor outcome. a The expression level of PDL1 was determined by qRT-PCR and Western blotting in seven different mammary cell lines, including one HME cell line (MCF-10A) and six TNBC cell lines. PDL1 expression was normalized using β-actin expression. b The expression level of PDL1 in 20 pairs of TNBC tissues and their matched normal adjacent tissues. c OS (left) and DFS (right) curves for breast cancer patients with positive or negative PDL1 expression. d OS (left) and DFS (right) curves for TNBC patients with positive or negative PDL1 expression. All the data are shown as the mean ± s.e.m. * P

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

PDL1 and LDHA act as ceRNAs in TNBC by regulating miR-34a. a The expression level of miR-34a was determined by qRT-PCR in stable cell lines expressing the PDL1 3’UTR or LDHA 3’UTR. U6 snRNA was used as an internal control. b The expression level of PDL1 was determined by qRT-PCR and Western blotting. β-Actin was used as an internal control. c The expression level of LDHA was determined by qRT-PCR and Western blotting. β-Actin was used as an internal control. d The expression level of LDHA was determined by Western blotting in xenograft tumors (six in each group). β-Actin was used as an internal control. e The expression level of PDL1 was determined by Western blotting in xenograft tumors (six in each group). β-Actin was used as an internal control. f Lactate production and glucose consumption was evaluated by measuring the lactate and glucose levels in cell medium. g The impact of the LDHA 3’UTR on immune cell populations in the tumor microenvironment. Flow cytometry revealed that the LDHA 3’UTR increased the number of macrophages and Tregs and reduced the number of CD8+ cells and CD4+ cells. All of the data are shown as the mean ± s.e.m. ** P
Figure Legend Snippet: PDL1 and LDHA act as ceRNAs in TNBC by regulating miR-34a. a The expression level of miR-34a was determined by qRT-PCR in stable cell lines expressing the PDL1 3’UTR or LDHA 3’UTR. U6 snRNA was used as an internal control. b The expression level of PDL1 was determined by qRT-PCR and Western blotting. β-Actin was used as an internal control. c The expression level of LDHA was determined by qRT-PCR and Western blotting. β-Actin was used as an internal control. d The expression level of LDHA was determined by Western blotting in xenograft tumors (six in each group). β-Actin was used as an internal control. e The expression level of PDL1 was determined by Western blotting in xenograft tumors (six in each group). β-Actin was used as an internal control. f Lactate production and glucose consumption was evaluated by measuring the lactate and glucose levels in cell medium. g The impact of the LDHA 3’UTR on immune cell populations in the tumor microenvironment. Flow cytometry revealed that the LDHA 3’UTR increased the number of macrophages and Tregs and reduced the number of CD8+ cells and CD4+ cells. All of the data are shown as the mean ± s.e.m. ** P

Techniques Used: Activated Clotting Time Assay, Expressing, Quantitative RT-PCR, Stable Transfection, Western Blot, Flow Cytometry, Cytometry

4) Product Images from "MicroRNA-16 is putatively involved in the NF-κB pathway regulation in ulcerative colitis through adenosine A2a receptor (A2aAR) mRNA targeting"

Article Title: MicroRNA-16 is putatively involved in the NF-κB pathway regulation in ulcerative colitis through adenosine A2a receptor (A2aAR) mRNA targeting

Journal: Scientific Reports

doi: 10.1038/srep30824

miR-16 regulates the endogenous expression of A2aAR. ( A ) qRT-PCR detected enhanced expression of miR-16 in a dose-dependent manner ( P
Figure Legend Snippet: miR-16 regulates the endogenous expression of A2aAR. ( A ) qRT-PCR detected enhanced expression of miR-16 in a dose-dependent manner ( P

Techniques Used: Expressing, Quantitative RT-PCR

Effect of miR-16 on the activation of NF-κB p65 and expression of IFN-γ and IL-8. HT-29 cells were transfected with 150 nM of miR-16 mimics, mimics-NC, miR-16 inhibitor and/or inhibitor-NC, respectively. These cells were then treated with TNF-α. ( A–C ) NF-κB p65 protein in cytosolic and nuclear fraction of these cells as analysed by western blot and quantified. Compared to the corresponding NC control, nuclear translocation of NF-κB p65 protein was enhanced in cells transfected with miR-16 mimics, and decreased in cells transfected with miR-16 inhibitor. ( D,E ) qRT-PCR detecting the expression of IFN-γ and IL-8 mRNAs in these cells. ( F,G ) ELISA for IFN-γ and IL-8 in cell supernatants. Data are presented as mean ± SD, * P
Figure Legend Snippet: Effect of miR-16 on the activation of NF-κB p65 and expression of IFN-γ and IL-8. HT-29 cells were transfected with 150 nM of miR-16 mimics, mimics-NC, miR-16 inhibitor and/or inhibitor-NC, respectively. These cells were then treated with TNF-α. ( A–C ) NF-κB p65 protein in cytosolic and nuclear fraction of these cells as analysed by western blot and quantified. Compared to the corresponding NC control, nuclear translocation of NF-κB p65 protein was enhanced in cells transfected with miR-16 mimics, and decreased in cells transfected with miR-16 inhibitor. ( D,E ) qRT-PCR detecting the expression of IFN-γ and IL-8 mRNAs in these cells. ( F,G ) ELISA for IFN-γ and IL-8 in cell supernatants. Data are presented as mean ± SD, * P

Techniques Used: Activation Assay, Expressing, Transfection, Western Blot, Translocation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Effect of TNF-α stimulation on NF-κB p65 activation and IFN-γ and IL-8 expression. HT-29 cells were treated with or without TNF-α for 12 h or 24 h. ( A–C ) NF-κB p65 protein in cytosolic and nuclear fraction of these cells were analysed by western blot, and quantified by Quantity One software. ( D,E ) IFN-γ and IL-8 mRNA expression in HT-29 cells detected by qRT-PCR. ( F,G ) ELISA for IFN-γ and IL-8 in supernatants released by these cells. Data are presented as mean ± SD, * P
Figure Legend Snippet: Effect of TNF-α stimulation on NF-κB p65 activation and IFN-γ and IL-8 expression. HT-29 cells were treated with or without TNF-α for 12 h or 24 h. ( A–C ) NF-κB p65 protein in cytosolic and nuclear fraction of these cells were analysed by western blot, and quantified by Quantity One software. ( D,E ) IFN-γ and IL-8 mRNA expression in HT-29 cells detected by qRT-PCR. ( F,G ) ELISA for IFN-γ and IL-8 in supernatants released by these cells. Data are presented as mean ± SD, * P

Techniques Used: Activation Assay, Expressing, Western Blot, Software, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Effect of A2aAR-siRNA on NF-κB p65 activation and IFN-γ and IL-8 expression. HT-29 cells were co-transfected with 150 nM of miR-16 inhibitor and A2aAR-siRNA or siRNA-NC, and stimulated with TNF-α. ( A–C ) Western blot analyses and quantification of NF-κB p65 protein in cytosolic and nuclear fraction of cells. ( B ) Silencing of the A2aAR significantly decreased the NF-κB p65 protein expression in cytosolic extracts, ( C ) and increased the NF-κB p65 protein expression in nuclear extracts. ( D,E ) Expression of IFN-γ and IL-8 mRNAs in these cells as detected by qRT-PCR. ( F,G ) ELISA for IFN-γ and IL-8 present in cell supernatants. Data are presented as mean ± SD, * P
Figure Legend Snippet: Effect of A2aAR-siRNA on NF-κB p65 activation and IFN-γ and IL-8 expression. HT-29 cells were co-transfected with 150 nM of miR-16 inhibitor and A2aAR-siRNA or siRNA-NC, and stimulated with TNF-α. ( A–C ) Western blot analyses and quantification of NF-κB p65 protein in cytosolic and nuclear fraction of cells. ( B ) Silencing of the A2aAR significantly decreased the NF-κB p65 protein expression in cytosolic extracts, ( C ) and increased the NF-κB p65 protein expression in nuclear extracts. ( D,E ) Expression of IFN-γ and IL-8 mRNAs in these cells as detected by qRT-PCR. ( F,G ) ELISA for IFN-γ and IL-8 present in cell supernatants. Data are presented as mean ± SD, * P

Techniques Used: Activation Assay, Expressing, Transfection, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Expression of the A2aAR and miR-16 in tissues. ( A ) IF staining of sigmoid biopsy section using specific antibody to label A2aAR protein and DAPI for counterstaining the nuclei (Blue). The strong green fluorescence signal indicating A2aAR was observed mainly in the sigmoid colonic epithelial cells. The expression of A2aAR was reduced in UC inflamed tissues. ( B ) miR-16 expression, and ( C ) A2aAR mRNA expression in individual sigmoid colon tissues (normal controls n = 20, IBS n = 22 and active UC n = 28), were measured by qRT-PCR (Data are shown as mean ± SD, ** P
Figure Legend Snippet: Expression of the A2aAR and miR-16 in tissues. ( A ) IF staining of sigmoid biopsy section using specific antibody to label A2aAR protein and DAPI for counterstaining the nuclei (Blue). The strong green fluorescence signal indicating A2aAR was observed mainly in the sigmoid colonic epithelial cells. The expression of A2aAR was reduced in UC inflamed tissues. ( B ) miR-16 expression, and ( C ) A2aAR mRNA expression in individual sigmoid colon tissues (normal controls n = 20, IBS n = 22 and active UC n = 28), were measured by qRT-PCR (Data are shown as mean ± SD, ** P

Techniques Used: Expressing, Staining, Fluorescence, Quantitative RT-PCR

5) Product Images from "MicroRNA-186-5p Inhibits Proliferation And Metastasis Of Esophageal Cancer By Mediating HOXA9"

Article Title: MicroRNA-186-5p Inhibits Proliferation And Metastasis Of Esophageal Cancer By Mediating HOXA9

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S227920

MiR-186-5p is underexpressed in esophageal cancer tissues and cell lines. ( A ) qRT-PCR was used to detect the difference in expression of miR-186-5p in esophageal cancer tumor tissues and adjacent tissues; ( B ) qRT-PCR was used to detect the expression level of miR-186-5p in esophageal cancer cell lines; ( C ) Kaplan Meier survival curve of esophageal cancer patients based on miR-186-5p expression; the prognosis of patients with low expression was significantly worse than that of high expression group. Data are mean ± SD, * P
Figure Legend Snippet: MiR-186-5p is underexpressed in esophageal cancer tissues and cell lines. ( A ) qRT-PCR was used to detect the difference in expression of miR-186-5p in esophageal cancer tumor tissues and adjacent tissues; ( B ) qRT-PCR was used to detect the expression level of miR-186-5p in esophageal cancer cell lines; ( C ) Kaplan Meier survival curve of esophageal cancer patients based on miR-186-5p expression; the prognosis of patients with low expression was significantly worse than that of high expression group. Data are mean ± SD, * P

Techniques Used: Quantitative RT-PCR, Expressing

Inhibition of esophageal cancer cell proliferation after overexpression of miR-186-5p. ( A ) qRT-PCR verified the interference efficiency of miR-186-5p after transfection of the miR-186-5p vector in the TE-1 and EC-109 cell lines; ( B ) The CCK-8 assay detects the effect of overexpression of the miR-186-5p vector on proliferation of esophageal cancer cells in TE-1 and EC-109 cell lines; ( C ) Plate cloning experiments were performed to detect the number of esophageal cancer proliferating positive cells after overexpression of miR-186-5p vector in TE-1 and EC-109 cell lines; the data were mean ± SD, * P
Figure Legend Snippet: Inhibition of esophageal cancer cell proliferation after overexpression of miR-186-5p. ( A ) qRT-PCR verified the interference efficiency of miR-186-5p after transfection of the miR-186-5p vector in the TE-1 and EC-109 cell lines; ( B ) The CCK-8 assay detects the effect of overexpression of the miR-186-5p vector on proliferation of esophageal cancer cells in TE-1 and EC-109 cell lines; ( C ) Plate cloning experiments were performed to detect the number of esophageal cancer proliferating positive cells after overexpression of miR-186-5p vector in TE-1 and EC-109 cell lines; the data were mean ± SD, * P

Techniques Used: Inhibition, Over Expression, Quantitative RT-PCR, Transfection, Plasmid Preparation, CCK-8 Assay, Clone Assay

miR-186-5p binds directly to HOXA9. ( A ) The dual luciferase reporter assay validated the direct targeting of miR-186-5p with HOXA9; ( B ) qRT-PCR verified the expression level of HOXA9 after transfection of miR-186-5p in TE-1 and EC-109 cell lines; ( C ) qRT-PCR was used to detect the difference in expression of HOXA9 in tumor tissues and adjacent tissues of esophageal carcinoma; ( D ) qRT-PCR was used to detect the expression level of HOXA9 in esophageal cancer cell lines; ( E ) There was a significant negative correlation between miR-186-5p and HOXA9 expression in esophageal cancer tissues. Data are mean ± SD, * P
Figure Legend Snippet: miR-186-5p binds directly to HOXA9. ( A ) The dual luciferase reporter assay validated the direct targeting of miR-186-5p with HOXA9; ( B ) qRT-PCR verified the expression level of HOXA9 after transfection of miR-186-5p in TE-1 and EC-109 cell lines; ( C ) qRT-PCR was used to detect the difference in expression of HOXA9 in tumor tissues and adjacent tissues of esophageal carcinoma; ( D ) qRT-PCR was used to detect the expression level of HOXA9 in esophageal cancer cell lines; ( E ) There was a significant negative correlation between miR-186-5p and HOXA9 expression in esophageal cancer tissues. Data are mean ± SD, * P

Techniques Used: Luciferase, Reporter Assay, Quantitative RT-PCR, Expressing, Transfection

miR-186-5p regulates the mechanism of action of HOXA9 to inhibit the development of esophageal cancer. ( A ) HOXA9 expression levels in eR esophageal cancer TE-1 and EC-109 cell lines co-transfected with miR-186-5p and HOXA9 were detected by qRT-PCR; ( B ) The CCK-8 assay detects the effect of co-transfection of miR-186-5p and HOXA9 overexpression vectors on proliferation of esophageal cancer cells in TE-1 and EC-109 cell lines; ( C ) Plate cloning experiments were performed to detect the number of esophageal cancer proliferation-positive cells after co-transfection of miR-186-5p and HOXA9 overexpression vectors in TE-1 and EC-109 cell lines; ( D ) Transwell assay was used to detect the effect of co-transfection of miR-186-5p and HOXA9 overexpression vectors on the migration ability of esophageal cancer cells in TE-1 and EC-109 cell lines. Data are mean ± SD, * P
Figure Legend Snippet: miR-186-5p regulates the mechanism of action of HOXA9 to inhibit the development of esophageal cancer. ( A ) HOXA9 expression levels in eR esophageal cancer TE-1 and EC-109 cell lines co-transfected with miR-186-5p and HOXA9 were detected by qRT-PCR; ( B ) The CCK-8 assay detects the effect of co-transfection of miR-186-5p and HOXA9 overexpression vectors on proliferation of esophageal cancer cells in TE-1 and EC-109 cell lines; ( C ) Plate cloning experiments were performed to detect the number of esophageal cancer proliferation-positive cells after co-transfection of miR-186-5p and HOXA9 overexpression vectors in TE-1 and EC-109 cell lines; ( D ) Transwell assay was used to detect the effect of co-transfection of miR-186-5p and HOXA9 overexpression vectors on the migration ability of esophageal cancer cells in TE-1 and EC-109 cell lines. Data are mean ± SD, * P

Techniques Used: Expressing, Transfection, Quantitative RT-PCR, CCK-8 Assay, Cotransfection, Over Expression, Clone Assay, Transwell Assay, Migration

6) Product Images from "Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression"

Article Title: Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression

Journal: Molecular Cancer

doi: 10.1186/s12943-018-0836-7

AFAP1-AS1 could directly bind to EZH2. a After nuclear and cytosolic separation, RNA expression levels were measured by qRT-PCR. GAPDH was used as a cytosol marker and U6 was used as a nucleus marker. b RIPs experiments for EZH2, SUZ12 were performed and the coprecipitated RNA was subjected to qRT-PCR for AFAP1-AS1
Figure Legend Snippet: AFAP1-AS1 could directly bind to EZH2. a After nuclear and cytosolic separation, RNA expression levels were measured by qRT-PCR. GAPDH was used as a cytosol marker and U6 was used as a nucleus marker. b RIPs experiments for EZH2, SUZ12 were performed and the coprecipitated RNA was subjected to qRT-PCR for AFAP1-AS1

Techniques Used: RNA Expression, Quantitative RT-PCR, Marker

AFAP1-AS1 regulates NSCLC cell proliferation in vitro. a Analysis of AFAP1-AS1 expression levels in NSCLC cell lines compared with a normal bronchial epithelial cell line (16HBE) using qRT-PCR. b The relative expression levels of AFAP1-AS1 in A549 and SPCA1 cells transfected with si-NC or si-AFAP1-AS1 (si-AFAP1-AS1 1# and si-AFAP1-AS1 2#) were measured using qPCR. c MTT assays were performed after transfection to determine the cell viability of the transfected A549 and SPCA1 cells. d Colony-forming assays were conducted after transfection to determine the proliferation of the transfected A549 and SPCA1 cells. The colonies were counted and captured. e BrdU assays were used to detect the cell proliferation after transfection, respectively. f After transfection, the cell cycle stages of A549 and SPCA1 cells were analyzed by flow cytometry. The bar chart represents the percentages of cells in the G1/G0, S, or G2/M phases, as indicated.*, P
Figure Legend Snippet: AFAP1-AS1 regulates NSCLC cell proliferation in vitro. a Analysis of AFAP1-AS1 expression levels in NSCLC cell lines compared with a normal bronchial epithelial cell line (16HBE) using qRT-PCR. b The relative expression levels of AFAP1-AS1 in A549 and SPCA1 cells transfected with si-NC or si-AFAP1-AS1 (si-AFAP1-AS1 1# and si-AFAP1-AS1 2#) were measured using qPCR. c MTT assays were performed after transfection to determine the cell viability of the transfected A549 and SPCA1 cells. d Colony-forming assays were conducted after transfection to determine the proliferation of the transfected A549 and SPCA1 cells. The colonies were counted and captured. e BrdU assays were used to detect the cell proliferation after transfection, respectively. f After transfection, the cell cycle stages of A549 and SPCA1 cells were analyzed by flow cytometry. The bar chart represents the percentages of cells in the G1/G0, S, or G2/M phases, as indicated.*, P

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, Real-time Polymerase Chain Reaction, MTT Assay, Flow Cytometry, Cytometry

RNA-seq assay after AFAP1-AS1 knockdown in A549 cells. a Mean-centered, hierarchical clustering of 591 transcripts altered (≥2-fold change) in si-NC-treated cells and siRNA-AFAP1-AS1 treated A549 cells. b Gene Ontology analysis for all genes with altered expressions. c The altered mRNA levels of genes were selectively confirmed by qRT-PCR in knockdown AFAP1-AS1
Figure Legend Snippet: RNA-seq assay after AFAP1-AS1 knockdown in A549 cells. a Mean-centered, hierarchical clustering of 591 transcripts altered (≥2-fold change) in si-NC-treated cells and siRNA-AFAP1-AS1 treated A549 cells. b Gene Ontology analysis for all genes with altered expressions. c The altered mRNA levels of genes were selectively confirmed by qRT-PCR in knockdown AFAP1-AS1

Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR

7) Product Images from "Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression"

Article Title: Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression

Journal: Molecular Cancer

doi: 10.1186/s12943-018-0836-7

AFAP1-AS1 could directly bind to EZH2. a After nuclear and cytosolic separation, RNA expression levels were measured by qRT-PCR. GAPDH was used as a cytosol marker and U6 was used as a nucleus marker. b RIPs experiments for EZH2, SUZ12 were performed and the coprecipitated RNA was subjected to qRT-PCR for AFAP1-AS1
Figure Legend Snippet: AFAP1-AS1 could directly bind to EZH2. a After nuclear and cytosolic separation, RNA expression levels were measured by qRT-PCR. GAPDH was used as a cytosol marker and U6 was used as a nucleus marker. b RIPs experiments for EZH2, SUZ12 were performed and the coprecipitated RNA was subjected to qRT-PCR for AFAP1-AS1

Techniques Used: RNA Expression, Quantitative RT-PCR, Marker

AFAP1-AS1 regulates NSCLC cell proliferation in vitro. a Analysis of AFAP1-AS1 expression levels in NSCLC cell lines compared with a normal bronchial epithelial cell line (16HBE) using qRT-PCR. b The relative expression levels of AFAP1-AS1 in A549 and SPCA1 cells transfected with si-NC or si-AFAP1-AS1 (si-AFAP1-AS1 1# and si-AFAP1-AS1 2#) were measured using qPCR. c MTT assays were performed after transfection to determine the cell viability of the transfected A549 and SPCA1 cells. d Colony-forming assays were conducted after transfection to determine the proliferation of the transfected A549 and SPCA1 cells. The colonies were counted and captured. e BrdU assays were used to detect the cell proliferation after transfection, respectively. f After transfection, the cell cycle stages of A549 and SPCA1 cells were analyzed by flow cytometry. The bar chart represents the percentages of cells in the G1/G0, S, or G2/M phases, as indicated.*, P
Figure Legend Snippet: AFAP1-AS1 regulates NSCLC cell proliferation in vitro. a Analysis of AFAP1-AS1 expression levels in NSCLC cell lines compared with a normal bronchial epithelial cell line (16HBE) using qRT-PCR. b The relative expression levels of AFAP1-AS1 in A549 and SPCA1 cells transfected with si-NC or si-AFAP1-AS1 (si-AFAP1-AS1 1# and si-AFAP1-AS1 2#) were measured using qPCR. c MTT assays were performed after transfection to determine the cell viability of the transfected A549 and SPCA1 cells. d Colony-forming assays were conducted after transfection to determine the proliferation of the transfected A549 and SPCA1 cells. The colonies were counted and captured. e BrdU assays were used to detect the cell proliferation after transfection, respectively. f After transfection, the cell cycle stages of A549 and SPCA1 cells were analyzed by flow cytometry. The bar chart represents the percentages of cells in the G1/G0, S, or G2/M phases, as indicated.*, P

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, Real-time Polymerase Chain Reaction, MTT Assay, Flow Cytometry, Cytometry

RNA-seq assay after AFAP1-AS1 knockdown in A549 cells. a Mean-centered, hierarchical clustering of 591 transcripts altered (≥2-fold change) in si-NC-treated cells and siRNA-AFAP1-AS1 treated A549 cells. b Gene Ontology analysis for all genes with altered expressions. c The altered mRNA levels of genes were selectively confirmed by qRT-PCR in knockdown AFAP1-AS1
Figure Legend Snippet: RNA-seq assay after AFAP1-AS1 knockdown in A549 cells. a Mean-centered, hierarchical clustering of 591 transcripts altered (≥2-fold change) in si-NC-treated cells and siRNA-AFAP1-AS1 treated A549 cells. b Gene Ontology analysis for all genes with altered expressions. c The altered mRNA levels of genes were selectively confirmed by qRT-PCR in knockdown AFAP1-AS1

Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR

8) Product Images from "Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma"

Article Title: Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma

Journal: BioMed Research International

doi: 10.1155/2013/251098

Degradation of matrix in situ is suppressed by knockdown of HOTAIR. (a) qRT-PCR analysis was performed to examine HOTAIR RNA levels in A375 cells transfected with siControl or siHOTAIR (I and II). (b) A375 cells were transfected with siControl or siHOTAIR (I and II) for 24 hours. In situ zymography was performed with Oregon green 488-conjugated gelatin. Cells were plated on gelatin matrix and incubated for 24 hours. The slides were observed by phase-contrast microscopy (left column), and gelatin degradation was visualized by fluorescence microscopy (right column).
Figure Legend Snippet: Degradation of matrix in situ is suppressed by knockdown of HOTAIR. (a) qRT-PCR analysis was performed to examine HOTAIR RNA levels in A375 cells transfected with siControl or siHOTAIR (I and II). (b) A375 cells were transfected with siControl or siHOTAIR (I and II) for 24 hours. In situ zymography was performed with Oregon green 488-conjugated gelatin. Cells were plated on gelatin matrix and incubated for 24 hours. The slides were observed by phase-contrast microscopy (left column), and gelatin degradation was visualized by fluorescence microscopy (right column).

Techniques Used: In Situ, Quantitative RT-PCR, Transfection, Zymography, Incubation, Microscopy, Fluorescence

Knockdown of HOTAIR decreases melanoma A375 cell motility. (a) qRT-PCR analysis was performed to examine HOTAIR RNA levels in A375 cells transfected with siControl, siHOTAIR I, or siHOTAIR II. (b) Wounds were introduced by scratching confluent monolayers of A375 cells transfected with siControl, siHOTAIR I, or siHOTAIR II. Migration was monitored by light microscopy at 0 hours and 24 hours (upper panel). The widths of the gaps from 3 experiments were measured and the results are presented in a bar graph (lower panel). * P
Figure Legend Snippet: Knockdown of HOTAIR decreases melanoma A375 cell motility. (a) qRT-PCR analysis was performed to examine HOTAIR RNA levels in A375 cells transfected with siControl, siHOTAIR I, or siHOTAIR II. (b) Wounds were introduced by scratching confluent monolayers of A375 cells transfected with siControl, siHOTAIR I, or siHOTAIR II. Migration was monitored by light microscopy at 0 hours and 24 hours (upper panel). The widths of the gaps from 3 experiments were measured and the results are presented in a bar graph (lower panel). * P

Techniques Used: Quantitative RT-PCR, Transfection, Migration, Light Microscopy

9) Product Images from "TREK-1 mediates isoflurane-induced cytotoxicity in astrocytes"

Article Title: TREK-1 mediates isoflurane-induced cytotoxicity in astrocytes

Journal: BMC Anesthesiology

doi: 10.1186/s12871-017-0420-5

Regulation of astrocytic TREK-1 influenced the expression of BDNF in cultured astrocytes after isoflurane exposure. The TREK-1 ( a , N = 5) and BDNF ( b , N = 5) mRNA levels were analysed by qRT-PCR in TREK-1 overexpression and knock-down models. a and b were treated with different doses of isoflurane (0%, 2.4%, and 3.6%) for 9 h. ISO, isoflurane. Data are presented as relative to the values of 0% isoflurane in Con group. The data are presented as the mean ± SEM. * P
Figure Legend Snippet: Regulation of astrocytic TREK-1 influenced the expression of BDNF in cultured astrocytes after isoflurane exposure. The TREK-1 ( a , N = 5) and BDNF ( b , N = 5) mRNA levels were analysed by qRT-PCR in TREK-1 overexpression and knock-down models. a and b were treated with different doses of isoflurane (0%, 2.4%, and 3.6%) for 9 h. ISO, isoflurane. Data are presented as relative to the values of 0% isoflurane in Con group. The data are presented as the mean ± SEM. * P

Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR, Over Expression

The over-expression of TREK-1 aggravated isoflurane-induced astrocytic cytotoxicity. a Verification of the astrocytic TREK-1 over-expression and knockdown models by Western blotting ( N = 4) and qRT-PCR ( N = 5). b Astrocytic viabilities were detected in the TREK-1 over-expression and knockdown models using MTT assays after treatment with different isoflurane doses (0%, 2.4%, and 3.6%) with an exposure duration of 9 h ( N = 5). Data are presented as relative to Con group. c The cytotoxicity induced by isoflurane (2.4%) exposure for 9 h was verified via LDH assay ( N = 5). Data are presented as relative to Con group. d The percentages of TUNEL-positive cells after 9 h of isoflurane exposure (2.4%) were determined in the astrocytic TREK-1 over-expression and knockdown models ( N = 3). ISO, isoflurane. The data are presented as the mean ± SEM. * P
Figure Legend Snippet: The over-expression of TREK-1 aggravated isoflurane-induced astrocytic cytotoxicity. a Verification of the astrocytic TREK-1 over-expression and knockdown models by Western blotting ( N = 4) and qRT-PCR ( N = 5). b Astrocytic viabilities were detected in the TREK-1 over-expression and knockdown models using MTT assays after treatment with different isoflurane doses (0%, 2.4%, and 3.6%) with an exposure duration of 9 h ( N = 5). Data are presented as relative to Con group. c The cytotoxicity induced by isoflurane (2.4%) exposure for 9 h was verified via LDH assay ( N = 5). Data are presented as relative to Con group. d The percentages of TUNEL-positive cells after 9 h of isoflurane exposure (2.4%) were determined in the astrocytic TREK-1 over-expression and knockdown models ( N = 3). ISO, isoflurane. The data are presented as the mean ± SEM. * P

Techniques Used: Over Expression, Western Blot, Quantitative RT-PCR, MTT Assay, Lactate Dehydrogenase Assay, TUNEL Assay

10) Product Images from "GSK3 Inhibitor-BIO Regulates Proliferation of Immortalized Pancreatic Mesenchymal Stem Cells (iPMSCs)"

Article Title: GSK3 Inhibitor-BIO Regulates Proliferation of Immortalized Pancreatic Mesenchymal Stem Cells (iPMSCs)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0031502

Potential differentiation of islet cells. A, The induced clusters treated with BIO or not were transferred into plates and stained positive for PDX1, C-peptide and insulin analysed by immunofluorescent staining; B, The specific markers of pancreatic islets including PDX1, Ngn3, Mafa, Glut2, PC1/3 and Insulin expressed in the iPMSCs treated with BIO or not by QRT-PCR; C, Glucose-stimulated insulin release and insulin content analysis. Bar = 20 µm, **P
Figure Legend Snippet: Potential differentiation of islet cells. A, The induced clusters treated with BIO or not were transferred into plates and stained positive for PDX1, C-peptide and insulin analysed by immunofluorescent staining; B, The specific markers of pancreatic islets including PDX1, Ngn3, Mafa, Glut2, PC1/3 and Insulin expressed in the iPMSCs treated with BIO or not by QRT-PCR; C, Glucose-stimulated insulin release and insulin content analysis. Bar = 20 µm, **P

Techniques Used: Staining, Quantitative RT-PCR

The GSK-3 inhibitor BIO promotes self-renewal of iPMSCs. A, Immunofluorescene staining analysis showed iPMSCs were positive for PDX1, Glut2, PCNA, C-Myc and TERT in the absence or presence of BIO, Bar = 20 µm; B, The percentage of positive cells treated with BIO or not from A; C, QRT-PCR analysis showed that the expression of pancreatic islets-specific marker PDX1 and pluripotent markers C-Myc, PCNA and TERT had an up-regulated trend in BIO cultured cells (24, 48, 72 h); D, Treated with BIO (24, 48, 72 h), western blotting analysis showed the expression of PDX1, PCNA, C-Myc, TERT and E-cadherin proteins had an upward trend compared with untreated cells. Bar = 20 µm, * P
Figure Legend Snippet: The GSK-3 inhibitor BIO promotes self-renewal of iPMSCs. A, Immunofluorescene staining analysis showed iPMSCs were positive for PDX1, Glut2, PCNA, C-Myc and TERT in the absence or presence of BIO, Bar = 20 µm; B, The percentage of positive cells treated with BIO or not from A; C, QRT-PCR analysis showed that the expression of pancreatic islets-specific marker PDX1 and pluripotent markers C-Myc, PCNA and TERT had an up-regulated trend in BIO cultured cells (24, 48, 72 h); D, Treated with BIO (24, 48, 72 h), western blotting analysis showed the expression of PDX1, PCNA, C-Myc, TERT and E-cadherin proteins had an upward trend compared with untreated cells. Bar = 20 µm, * P

Techniques Used: Staining, Quantitative RT-PCR, Expressing, Marker, Cell Culture, Western Blot

11) Product Images from "Transcriptomic analysis of the prothoracic gland from two lepidopteran insects, domesticated silkmoth Bombyx mori and wild silkmoth Antheraea pernyi"

Article Title: Transcriptomic analysis of the prothoracic gland from two lepidopteran insects, domesticated silkmoth Bombyx mori and wild silkmoth Antheraea pernyi

Journal: Scientific Reports

doi: 10.1038/s41598-019-41864-0

qRT-PCR results of expression of the JH related genes. The expression patterns of 8 genes related to JH in B. mori PG during development in whole fifth larval instar. The relative expression levels were normalized to the Bmrp49 levels. The values are the mean ± SEM (n = 3) of three repeat experiments using qRT-PCR.
Figure Legend Snippet: qRT-PCR results of expression of the JH related genes. The expression patterns of 8 genes related to JH in B. mori PG during development in whole fifth larval instar. The relative expression levels were normalized to the Bmrp49 levels. The values are the mean ± SEM (n = 3) of three repeat experiments using qRT-PCR.

Techniques Used: Quantitative RT-PCR, Expressing

qRT-PCR results of expression of the clock genes. The expression patterns of 12 genes related to clock in B. mori PG during development in whole fifth larval instar. The relative expression levels were normalized to the Bmrp49 levels. The values are the mean ± SEM (n = 3) of three repeat experiments using qRT-PCR.
Figure Legend Snippet: qRT-PCR results of expression of the clock genes. The expression patterns of 12 genes related to clock in B. mori PG during development in whole fifth larval instar. The relative expression levels were normalized to the Bmrp49 levels. The values are the mean ± SEM (n = 3) of three repeat experiments using qRT-PCR.

Techniques Used: Quantitative RT-PCR, Expressing

12) Product Images from "A transcriptome study on Macrobrachium nipponense hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)"

Article Title: A transcriptome study on Macrobrachium nipponense hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200222

Comparison of the expression profiles of selected genes as determined by RNA-Seq (Illumina Hiseq2500 TM sequencing) and qRT-PCR. The letter “A” and “B” in the RNA-Seq and qRT-PCR mean the comparison of expression profiles of selected genes between control and moribund samples (the correlation coefficient was 0.92), the comparison of expression profiles of selected genes between control and survived samples (the correlation coefficient was 0.95), respectively. Target gene abbreviations are as follows: TSPAN8 –TSPAN8, DPAGT1 –UDP-N-acetylglucosamine—dolichyl-phosphate N-acetylglucosaminephosphotransferase, Gag-pol –Gag-Pol polyprotein, ARSB–arylsulfatase B, MKK2 –MAP kinase-activated protein kinase 2, PDK–pyruvate dehydrogenase (acetyl-transferring) kinase, ILK–integrin-linked protein kinase, AP–acid phosphatase, ERI3 –ERI1 exoribonuclease 3, ZMYM1 –zinc finger MYM-type protein 1. Error bars indicated standard deviations of averages from three replicates.
Figure Legend Snippet: Comparison of the expression profiles of selected genes as determined by RNA-Seq (Illumina Hiseq2500 TM sequencing) and qRT-PCR. The letter “A” and “B” in the RNA-Seq and qRT-PCR mean the comparison of expression profiles of selected genes between control and moribund samples (the correlation coefficient was 0.92), the comparison of expression profiles of selected genes between control and survived samples (the correlation coefficient was 0.95), respectively. Target gene abbreviations are as follows: TSPAN8 –TSPAN8, DPAGT1 –UDP-N-acetylglucosamine—dolichyl-phosphate N-acetylglucosaminephosphotransferase, Gag-pol –Gag-Pol polyprotein, ARSB–arylsulfatase B, MKK2 –MAP kinase-activated protein kinase 2, PDK–pyruvate dehydrogenase (acetyl-transferring) kinase, ILK–integrin-linked protein kinase, AP–acid phosphatase, ERI3 –ERI1 exoribonuclease 3, ZMYM1 –zinc finger MYM-type protein 1. Error bars indicated standard deviations of averages from three replicates.

Techniques Used: Expressing, RNA Sequencing Assay, Sequencing, Quantitative RT-PCR, Transferring

13) Product Images from "Tomato lncRNA23468 functions as a competing endogenous RNA to modulate NBS-LRR genes by decoying miR482b in the tomato-Phytophthora infestans interaction"

Article Title: Tomato lncRNA23468 functions as a competing endogenous RNA to modulate NBS-LRR genes by decoying miR482b in the tomato-Phytophthora infestans interaction

Journal: Horticulture Research

doi: 10.1038/s41438-018-0096-0

lncRNA23468 functions to modulate NBS-LRR genes by decoying miR482b. a Schematic diagram of the gene cassette containing lncRNA23468 and mlncRNA23468. b qRT-PCR analysis of lncRNA23468, miR482b and the target genes in the EV, mOE23468 and OE23468 tomato plants. c Disease signs on the detached leaves from the EV, mOE23468 and OE23468 tomato plants at 5 dpi. Scale bars = 0.5 cm. d The diameter of the lesion of the detached leaves from EV, mOE23468 and OE23468 tomato plants at 5 dpi. All data are the means ± SE of three independent experiments. Different letters among the groups indicate a significant difference at the P = 0.05 level
Figure Legend Snippet: lncRNA23468 functions to modulate NBS-LRR genes by decoying miR482b. a Schematic diagram of the gene cassette containing lncRNA23468 and mlncRNA23468. b qRT-PCR analysis of lncRNA23468, miR482b and the target genes in the EV, mOE23468 and OE23468 tomato plants. c Disease signs on the detached leaves from the EV, mOE23468 and OE23468 tomato plants at 5 dpi. Scale bars = 0.5 cm. d The diameter of the lesion of the detached leaves from EV, mOE23468 and OE23468 tomato plants at 5 dpi. All data are the means ± SE of three independent experiments. Different letters among the groups indicate a significant difference at the P = 0.05 level

Techniques Used: Quantitative RT-PCR

14) Product Images from "Transcriptome analysis of Cucumis sativus infected by Cucurbit chlorotic yellows virus"

Article Title: Transcriptome analysis of Cucumis sativus infected by Cucurbit chlorotic yellows virus

Journal: Virology Journal

doi: 10.1186/s12985-017-0690-z

Quantitative real-time PCR (RT-PCR) validation of DEGs. The cucumber ubiquitin gene was used as an internal control. Error bars represent the standard deviation of the qRT-PCR signals ( n = 3). Asterisks indicate statistically significant differences compared with the control ( P
Figure Legend Snippet: Quantitative real-time PCR (RT-PCR) validation of DEGs. The cucumber ubiquitin gene was used as an internal control. Error bars represent the standard deviation of the qRT-PCR signals ( n = 3). Asterisks indicate statistically significant differences compared with the control ( P

Techniques Used: Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Quantitative RT-PCR

15) Product Images from "Quantitative Trait Locus Mapping and Candidate Gene Analysis for Verticillium Wilt Resistance Using Gossypium barbadense Chromosomal Segment Introgressed Line"

Article Title: Quantitative Trait Locus Mapping and Candidate Gene Analysis for Verticillium Wilt Resistance Using Gossypium barbadense Chromosomal Segment Introgressed Line

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.00682

Resistance phenotypes and detection of V. dahliae biomass of two parents, CSILSuVR043 and Sumian 8, after artificial VW inoculation in 2014 in Nanjing. (A) The resistance phenotype of CSILSuVR043 and Sumian 8. The picture was taken 4 weeks after inoculation. (B) Detection of V. dahliae biomass in CSILSuVR043, Sumian 8 and H7124 using qRT-PCR. DNA was extracted from the lower 2 cm of stems 21 days after inoculation with V. dahliae . The relative average fungal biomass is shown with standard errors. The letter indicates significant differences according to Duncan's multiple range tests ( p
Figure Legend Snippet: Resistance phenotypes and detection of V. dahliae biomass of two parents, CSILSuVR043 and Sumian 8, after artificial VW inoculation in 2014 in Nanjing. (A) The resistance phenotype of CSILSuVR043 and Sumian 8. The picture was taken 4 weeks after inoculation. (B) Detection of V. dahliae biomass in CSILSuVR043, Sumian 8 and H7124 using qRT-PCR. DNA was extracted from the lower 2 cm of stems 21 days after inoculation with V. dahliae . The relative average fungal biomass is shown with standard errors. The letter indicates significant differences according to Duncan's multiple range tests ( p

Techniques Used: Quantitative RT-PCR

16) Product Images from "Characterization of Conserved and Novel microRNAs in Lilium lancifolium Thunb. by High-Throughput Sequencing"

Article Title: Characterization of Conserved and Novel microRNAs in Lilium lancifolium Thunb. by High-Throughput Sequencing

Journal: Scientific Reports

doi: 10.1038/s41598-018-21193-4

Validation of miRNAs and targets using qRT-PCR. The X axis represents different tissues. The Y axis represents the relative expression level of miRNAs or targets.The amount of expression of miRNAs and targets was normalized to the level of 5.8S rRNA and 18S rRNA, respectively. Different letters indicate significant differences at P
Figure Legend Snippet: Validation of miRNAs and targets using qRT-PCR. The X axis represents different tissues. The Y axis represents the relative expression level of miRNAs or targets.The amount of expression of miRNAs and targets was normalized to the level of 5.8S rRNA and 18S rRNA, respectively. Different letters indicate significant differences at P

Techniques Used: Quantitative RT-PCR, Expressing

17) Product Images from "miR-30b, Down-Regulated in Gastric Cancer, Promotes Apoptosis and Suppresses Tumor Growth by Targeting Plasminogen Activator Inhibitor-1"

Article Title: miR-30b, Down-Regulated in Gastric Cancer, Promotes Apoptosis and Suppresses Tumor Growth by Targeting Plasminogen Activator Inhibitor-1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0106049

Inverse correlation between miR-30b and PAI-1 expression in gastric cancer cell lines and human tumor tissues. ( A ) The expression of mir-30b and PAI-1 in gastric cancer cell lines MKN45, MGC-823, SGC-7901, AGS and HGC-27. Top, western blot analysis of PAI-1 protein levels; bottom, qRT-PCR analysis of miR-30b levels. Data represent means±S.D. from three independent experiments. ( B ) miR-30b and PAI-1 mRNA levels in gastric cancer tissues were analyzed by qRT-PCR. The inset graph indicated a statistically significant inverse correlation ( R = −0.6475, P = 0.0123). U6 and β-actin served as internal normalized references for miR-30b and PAI-1 mRNA, respectively.
Figure Legend Snippet: Inverse correlation between miR-30b and PAI-1 expression in gastric cancer cell lines and human tumor tissues. ( A ) The expression of mir-30b and PAI-1 in gastric cancer cell lines MKN45, MGC-823, SGC-7901, AGS and HGC-27. Top, western blot analysis of PAI-1 protein levels; bottom, qRT-PCR analysis of miR-30b levels. Data represent means±S.D. from three independent experiments. ( B ) miR-30b and PAI-1 mRNA levels in gastric cancer tissues were analyzed by qRT-PCR. The inset graph indicated a statistically significant inverse correlation ( R = −0.6475, P = 0.0123). U6 and β-actin served as internal normalized references for miR-30b and PAI-1 mRNA, respectively.

Techniques Used: Expressing, Western Blot, Quantitative RT-PCR

18) Product Images from "Transcriptome Analysis by Illumina High-Throughout Paired-End Sequencing Reveals the Complexity of Differential Gene Expression during In Vitro Plantlet Growth and Flowering in Amaranthus tricolor L."

Article Title: Transcriptome Analysis by Illumina High-Throughout Paired-End Sequencing Reveals the Complexity of Differential Gene Expression during In Vitro Plantlet Growth and Flowering in Amaranthus tricolor L.

Journal: PLoS ONE

doi: 10.1371/journal.pone.0100919

Results of qRT-PCR analysis of 26 candidate unigenes during Am. tricolor in vitro plantlet growth and flowering. (a) Plant circadian rhythm pathway-related unigenes. (b) Ubiquitin-mediated proteolysis pathway-related unigenes. (c–d) Genes related to other or unknown pathways.
Figure Legend Snippet: Results of qRT-PCR analysis of 26 candidate unigenes during Am. tricolor in vitro plantlet growth and flowering. (a) Plant circadian rhythm pathway-related unigenes. (b) Ubiquitin-mediated proteolysis pathway-related unigenes. (c–d) Genes related to other or unknown pathways.

Techniques Used: Quantitative RT-PCR, In Vitro

19) Product Images from "The long non-coding RNA lncRNA973 is involved in cotton response to salt stress"

Article Title: The long non-coding RNA lncRNA973 is involved in cotton response to salt stress

Journal: BMC Plant Biology

doi: 10.1186/s12870-019-2088-0

Characteristics of lncRNA973. a . Schematic diagram of the location of lncRNA973 in a chromosome. Yellow boxes show the exon of lncRNA973. P1 and P2 indicated the primers used for qRT-PCR to detect the expression level of lncRNA973 X1 and lncRNA973 X2 , respectively. b . Expression levels of lncRNA973 transcripts in cotton stem, root and cotyledon, determined by qPCR. c . Expression levels of lncRNA973 X1 and X2 in cotton true leaves treated with 250 mM NaCl at different time points, determined by qPCR. The lncRNA973 expression level was normalized by the GhUBQ7 expression level. The relative expression levels of lncRNA973 in stem and NaCl-0 h were assigned a value of 1, respectively. Error bars indicate ±SD of three biological replicates, with each measured in triplicate. Samples marked with different letters show a significant difference at p
Figure Legend Snippet: Characteristics of lncRNA973. a . Schematic diagram of the location of lncRNA973 in a chromosome. Yellow boxes show the exon of lncRNA973. P1 and P2 indicated the primers used for qRT-PCR to detect the expression level of lncRNA973 X1 and lncRNA973 X2 , respectively. b . Expression levels of lncRNA973 transcripts in cotton stem, root and cotyledon, determined by qPCR. c . Expression levels of lncRNA973 X1 and X2 in cotton true leaves treated with 250 mM NaCl at different time points, determined by qPCR. The lncRNA973 expression level was normalized by the GhUBQ7 expression level. The relative expression levels of lncRNA973 in stem and NaCl-0 h were assigned a value of 1, respectively. Error bars indicate ±SD of three biological replicates, with each measured in triplicate. Samples marked with different letters show a significant difference at p

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

20) Product Images from "Mitochondrial-Nuclear DNA Interactions Contribute to the Regulation of Nuclear Transcript Levels as Part of the Inter-Organelle Communication System"

Article Title: Mitochondrial-Nuclear DNA Interactions Contribute to the Regulation of Nuclear Transcript Levels as Part of the Inter-Organelle Communication System

Journal: PLoS ONE

doi: 10.1371/journal.pone.0030943

Knocking out mitochondrial encoded reverse transcriptase activity results in increased transcript levels of nuclear genes that are involved in Mito-nDNA interactions. A) Nuclear encoded MSY1 transcript levels were determined by qRT-PCR in WT (strain 161-U7), 161-U7 GII-0 (lacks both the mitochondrial group II introns and the COX1 interacting region; Figure 4A ), and 161-U7 GII-0 a15γ (contains the interacting region and lacks the group II introns; Figure 4A ) cells. B) Nuclear encoded RSM7 transcript levels were determined by qRT-PCR in: WT (strain 161-U7); 161-U7 GII-0; and 161-U7 GII-0 a15γ cells. Neither 161-U7 GII-0 nor 161-U7 GII-0 a15γ has any alteration within the Q0182 open reading frame. C) Deletion of MRS1 (BY4741 Δ mrs1 ), a nuclear gene involved in splicing mitochondrial type-I introns, has no effect on i) MSY1 or ii) RSM7 transcript levels. All transcript levels were standardized to nuclear ACT1 and expressed as percentage of wild-type (set at 100%) +/− standard error of the mean (n = 2).
Figure Legend Snippet: Knocking out mitochondrial encoded reverse transcriptase activity results in increased transcript levels of nuclear genes that are involved in Mito-nDNA interactions. A) Nuclear encoded MSY1 transcript levels were determined by qRT-PCR in WT (strain 161-U7), 161-U7 GII-0 (lacks both the mitochondrial group II introns and the COX1 interacting region; Figure 4A ), and 161-U7 GII-0 a15γ (contains the interacting region and lacks the group II introns; Figure 4A ) cells. B) Nuclear encoded RSM7 transcript levels were determined by qRT-PCR in: WT (strain 161-U7); 161-U7 GII-0; and 161-U7 GII-0 a15γ cells. Neither 161-U7 GII-0 nor 161-U7 GII-0 a15γ has any alteration within the Q0182 open reading frame. C) Deletion of MRS1 (BY4741 Δ mrs1 ), a nuclear gene involved in splicing mitochondrial type-I introns, has no effect on i) MSY1 or ii) RSM7 transcript levels. All transcript levels were standardized to nuclear ACT1 and expressed as percentage of wild-type (set at 100%) +/− standard error of the mean (n = 2).

Techniques Used: Activity Assay, Quantitative RT-PCR

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RNA Extraction:

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RNA Sequencing Assay:

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Synthesized:

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Isolation:

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Transfection:

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Cell Culture:

Article Title: MicroRNA-16 is putatively involved in the NF-κB pathway regulation in ulcerative colitis through adenosine A2a receptor (A2aAR) mRNA targeting
Article Snippet: Real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) For qRT-PCR experiments, total RNA from colonic biopsies or cultured cells were extracted using RNAiso Plus according to the manufacturer’s instructions (Takara, Japan).The concentration and purity of the isolated total RNA samples were verified using an Eppendorf® BioPhotometer Plus (Eppendorf, Hamburg, Germany). .. The One Step PrimeScript® miRNA cDNA Synthesis Kit (Takara, Japan) and PrimeScript® RT Master Mix Perfect Real Time (Takara, Japan) were used to reverse-transcribe the miRNA and mRNA, respectively, according to the manufacturer’s instructions. qRT-PCR reactions were performed in triplicate using SYBR® Premix Ex TaqTM II (Perfect Real Time, Takara, Japan) on LightCycler® (Roche Diagnostics, Nutley, NJ, USA) in a 96-well format, over 45 cycles with denaturation at 95 °C for 10 s and annealing at 60 °C for 20 s. U6 small RNA was used to normalize the levels of miR-16, and GAPDH was used to normalize the mRNA expression levels of A2aAR, IFN-γ, and IL-8.

Article Title: Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression
Article Snippet: RNA extraction and qRT-PCR analyses The total RNA was extracted from tissues or cultured cells with TRIzol reagent (Invitrogen), according to the manufacturer’s protocol. .. One microgram total RNA was reverse transcribed using PrimeScript RT Reagent Kit with gDNA Eraser (Takara). cDNA was used for subsequent qRT-PCR reactions (SYBR, TaKaRa) according to the manufacturer’s instructions.

Article Title: MicroRNA-186-5p Inhibits Proliferation And Metastasis Of Esophageal Cancer By Mediating HOXA9
Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) The transfected cells were cultured for 48 h, and RNA was extracted and reverse transcribed into cDNA using Primescript RT Reagent (TaKaRa, Otsu, Japan). .. QRT-PCR reactions were performed using SYBR® Premix Ex TaqTM (TaKaRa, Otsu, Japan), and StepOne Plus Real-time PCR System (Applied BiPCaystems, FP Cater City, CA, USA).

Size-exclusion Chromatography:

Article Title: GSK3 Inhibitor-BIO Regulates Proliferation of Immortalized Pancreatic Mesenchymal Stem Cells (iPMSCs)
Article Snippet: QRT-PCR The QRT-PCR reactions were set up in 25 µL reaction mixtures containing 12.5 µL 1× SYBR@ PremixExTaqTM (TaKaRa, Biotech. .. The reaction conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec, and 58°C for 20 sec. All expression levels were normalized to β-actin in each well.

SYBR Green Assay:

Article Title: AVE0991, a nonpeptide analogue of Ang-(1-7), attenuates aging-related neuroinflammation
Article Snippet: .. After that, qRT-PCR reactions were performed with SYBR Green Premix Ex Taq (Takara Bio, Inc.) and specific primers (See ). .. Measurement of brain ACE2 activity ACE2 activity in mice brain was measured by a SensoLyte 390 ACE2 activity assay kit (AnaSpec) with Mc-Ala/Dnp fluorescence resonance energy transfer peptides as described [ ].

Article Title: Transcriptome analysis of Cucumis sativus infected by Cucurbit chlorotic yellows virus
Article Snippet: .. The qRT-PCR reactions were performed with 5 μL of SYBR Green master mix, 20 ng of cDNA, and 200 nM each of the sense and antisense primers, in a total volume of 10 μL (Takara). ..

Reverse Transcription Polymerase Chain Reaction:

Article Title: TREK-1 mediates isoflurane-induced cytotoxicity in astrocytes
Article Snippet: Paragraph title: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) ... Following reverse transcription, qRT-PCR reactions were performed with SYBR Premix Ex Taq (TaKaRa).

Random Hexamer Labeling:

Article Title: Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma
Article Snippet: Random hexamer primers were used in the RT reactions. .. GAPDH was used as an endogenous control for the qRT-PCR reactions (TaKaRa, Shiga, Japan).

Polymerase Chain Reaction:

Article Title: MicroRNA-16 is putatively involved in the NF-κB pathway regulation in ulcerative colitis through adenosine A2a receptor (A2aAR) mRNA targeting
Article Snippet: Paragraph title: Real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) ... The One Step PrimeScript® miRNA cDNA Synthesis Kit (Takara, Japan) and PrimeScript® RT Master Mix Perfect Real Time (Takara, Japan) were used to reverse-transcribe the miRNA and mRNA, respectively, according to the manufacturer’s instructions. qRT-PCR reactions were performed in triplicate using SYBR® Premix Ex TaqTM II (Perfect Real Time, Takara, Japan) on LightCycler® (Roche Diagnostics, Nutley, NJ, USA) in a 96-well format, over 45 cycles with denaturation at 95 °C for 10 s and annealing at 60 °C for 20 s. U6 small RNA was used to normalize the levels of miR-16, and GAPDH was used to normalize the mRNA expression levels of A2aAR, IFN-γ, and IL-8.

Article Title: Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma
Article Snippet: Paragraph title: 2.3. Quantitative Reverse Transcriptase PCR (qRT-PCR) of Six lncRNAs ... GAPDH was used as an endogenous control for the qRT-PCR reactions (TaKaRa, Shiga, Japan).

Article Title: AVE0991, a nonpeptide analogue of Ang-(1-7), attenuates aging-related neuroinflammation
Article Snippet: Paragraph title: Quantitative Reverse Transcription PCR (qRT-PCR) ... After that, qRT-PCR reactions were performed with SYBR Green Premix Ex Taq (Takara Bio, Inc.) and specific primers (See ).

Quantitative RT-PCR:

Article Title: MicroRNA-16 is putatively involved in the NF-κB pathway regulation in ulcerative colitis through adenosine A2a receptor (A2aAR) mRNA targeting
Article Snippet: .. The One Step PrimeScript® miRNA cDNA Synthesis Kit (Takara, Japan) and PrimeScript® RT Master Mix Perfect Real Time (Takara, Japan) were used to reverse-transcribe the miRNA and mRNA, respectively, according to the manufacturer’s instructions. qRT-PCR reactions were performed in triplicate using SYBR® Premix Ex TaqTM II (Perfect Real Time, Takara, Japan) on LightCycler® (Roche Diagnostics, Nutley, NJ, USA) in a 96-well format, over 45 cycles with denaturation at 95 °C for 10 s and annealing at 60 °C for 20 s. U6 small RNA was used to normalize the levels of miR-16, and GAPDH was used to normalize the mRNA expression levels of A2aAR, IFN-γ, and IL-8. .. The relative expression was calculated using the 2−∆CT method.

Article Title: Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression
Article Snippet: .. One microgram total RNA was reverse transcribed using PrimeScript RT Reagent Kit with gDNA Eraser (Takara). cDNA was used for subsequent qRT-PCR reactions (SYBR, TaKaRa) according to the manufacturer’s instructions. .. The results were normalized to the expression of GAPDH.

Article Title: PDL1 And LDHA act as ceRNAs in triple negative breast cancer by regulating miR-34a
Article Snippet: .. Reverse transcription and qRT-PCR reactions of mRNAs were performed using PrimeScript™ RT Master Mix and SYBR® Premix Ex TaqTM II (Takara, Japan). .. The primers for LDHA, PDL1 and β-actin were synthesized by Invitrogen: hsa-LDHA-forward, 5′-ATGGCAACTCTAAAGGATCAGC-3′; hsa-LDHA-reverse, 5′-CCAACCCCAACAACTGTAATCT-3′; mus-LDHA-forward, 5′-TGTCTCCAGCAAAGACTACTGT-3′; mus-LDHA-reverse, 5′-GACTGTACTTGACAATGTTGGGA-3′; hsa-PDL1-forward, 5′-GACATGTCAGGCTGAGGGCT-3′; hsa-PDL1-reverse, 5′-TGATTCTCAGTGTGCTGGTCACA-3′; mus-PDL1-forward, 5′-GCTCCAAAGGACTTGTACGTG-3′; mus-PDL1-reverse, 5′-TGATCTGAAGGGCAGCATTTC-3′; hsa-β-actin-forward, 5′-AGCGAGCATCCCCCAAAGTT-3′; hsa-β-actin-reverse, 5′-GGGCACGAAGGCTCATCATT-3′; mus-β-actin-forward, 5′-GTGACGTTGACATCCGTAAAGA-3′; and mus-β-actin-reverse, 5′-GCCGGACTCATCGTACTCC-3′.

Article Title: Tomato lncRNA23468 functions as a competing endogenous RNA to modulate NBS-LRR genes by decoying miR482b in the tomato-Phytophthora infestans interaction
Article Snippet: .. The qRT-PCR reactions were performed by using a SYBR Premix Ex TaqTM II kit (TaKaRa, Dalian, China). .. The qRT-PCR reactions of the selected genes, miRNAs and lncRNAs were performed on an ABI7500.

Article Title: MicroRNA-186-5p Inhibits Proliferation And Metastasis Of Esophageal Cancer By Mediating HOXA9
Article Snippet: .. QRT-PCR reactions were performed using SYBR® Premix Ex TaqTM (TaKaRa, Otsu, Japan), and StepOne Plus Real-time PCR System (Applied BiPCaystems, FP Cater City, CA, USA). .. The relative expression levels of microRNA-186-5p and HOXA9 were determined according to the methods described above.

Article Title: Transcriptomic analysis of the prothoracic gland from two lepidopteran insects, domesticated silkmoth Bombyx mori and wild silkmoth Antheraea pernyi
Article Snippet: .. The total volume of qRT-PCR reactions was 10 µl, containing 3.6 µl of TB Green Premix Ex Taq (TaKaRa), 0.4 µl of specific primers (10 µM), 1 µl of cDNA and 5 µl of ddH2 O. qRT-PCR was performed with a BIO-RAD CFX Connect Real-Time System, and the conditions were as follows: 95 °C for 30 s followed by 40 cycles in 95 °C for 5 s and 60 °C for 30 s. Gene-specific primers used for qRT-PCR analysis are listed in Additional file: Table . .. The mRNA expression levels of the genes of interest were calculated with the 2−ΔΔCt method and nomalized to the abundance of a house-keeping gene, ribosome protein 49 (rp49 ).

Article Title: Quantitative Trait Locus Mapping and Candidate Gene Analysis for Verticillium Wilt Resistance Using Gossypium barbadense Chromosomal Segment Introgressed Line
Article Snippet: .. All qRT-PCR reactions were performed on an ABI PRISM 7500 Real-Time PCR System using the SYBR Premix Ex Taq II kit (TaKaRa, Japan, Code No. RR820A) according to the manufacturer's instructions. .. Three biological replicates were conducted for each qRT-PCR reaction.

Article Title: Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma
Article Snippet: .. GAPDH was used as an endogenous control for the qRT-PCR reactions (TaKaRa, Shiga, Japan). .. The 2−ΔΔCt method was used to calculate the expression of each lncRNA.

Article Title: Heme acts through the Bach1b/Nrf2a-MafK pathway to regulate exocrine peptidase precursor genes in porphyric zebrafish
Article Snippet: .. RNA extraction and qRT-PCR Total RNA was extracted using the TRIzol® Reagent, according to the manufacturer’s instructions (Invitrogen). cDNA was synthesized by using reverse transcription with the M-MLV reverse transcription kit (Invitrogen), which was then used as the template for qRT-PCR analysis. qRT-PCR reactions were performed with the ABI StepOnePlus™ system, using SYBR® Premix Ex Taq™ (TaKaRa) and the following thermal profile: 95°C for 3 minutes; 95°C for 10 seconds; 58°C for 30 seconds for 40 cycles. ..

Article Title: AVE0991, a nonpeptide analogue of Ang-(1-7), attenuates aging-related neuroinflammation
Article Snippet: .. After that, qRT-PCR reactions were performed with SYBR Green Premix Ex Taq (Takara Bio, Inc.) and specific primers (See ). .. Measurement of brain ACE2 activity ACE2 activity in mice brain was measured by a SensoLyte 390 ACE2 activity assay kit (AnaSpec) with Mc-Ala/Dnp fluorescence resonance energy transfer peptides as described [ ].

Article Title: A transcriptome study on Macrobrachium nipponense hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)
Article Snippet: .. Optimized qRT-PCR reactions were performed following the manufacturer’s instructions (SYBR Premix Ex Taq, Takara Bio, Japan.) on a real-time thermal cycler (Bio-Rad, USA), using β-actin as a reference gene. .. Primers for qRT-PCR were carefully designed using Primer Premier 3 software and listed in .

Article Title: GSK3 Inhibitor-BIO Regulates Proliferation of Immortalized Pancreatic Mesenchymal Stem Cells (iPMSCs)
Article Snippet: .. QRT-PCR The QRT-PCR reactions were set up in 25 µL reaction mixtures containing 12.5 µL 1× SYBR@ PremixExTaqTM (TaKaRa, Biotech. ..

Article Title: TREK-1 mediates isoflurane-induced cytotoxicity in astrocytes
Article Snippet: .. Following reverse transcription, qRT-PCR reactions were performed with SYBR Premix Ex Taq (TaKaRa). ..

Expressing:

Article Title: MicroRNA-16 is putatively involved in the NF-κB pathway regulation in ulcerative colitis through adenosine A2a receptor (A2aAR) mRNA targeting
Article Snippet: .. The One Step PrimeScript® miRNA cDNA Synthesis Kit (Takara, Japan) and PrimeScript® RT Master Mix Perfect Real Time (Takara, Japan) were used to reverse-transcribe the miRNA and mRNA, respectively, according to the manufacturer’s instructions. qRT-PCR reactions were performed in triplicate using SYBR® Premix Ex TaqTM II (Perfect Real Time, Takara, Japan) on LightCycler® (Roche Diagnostics, Nutley, NJ, USA) in a 96-well format, over 45 cycles with denaturation at 95 °C for 10 s and annealing at 60 °C for 20 s. U6 small RNA was used to normalize the levels of miR-16, and GAPDH was used to normalize the mRNA expression levels of A2aAR, IFN-γ, and IL-8. .. The relative expression was calculated using the 2−∆CT method.

Article Title: Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression
Article Snippet: One microgram total RNA was reverse transcribed using PrimeScript RT Reagent Kit with gDNA Eraser (Takara). cDNA was used for subsequent qRT-PCR reactions (SYBR, TaKaRa) according to the manufacturer’s instructions. .. The results were normalized to the expression of GAPDH.

Article Title: MicroRNA-186-5p Inhibits Proliferation And Metastasis Of Esophageal Cancer By Mediating HOXA9
Article Snippet: QRT-PCR reactions were performed using SYBR® Premix Ex TaqTM (TaKaRa, Otsu, Japan), and StepOne Plus Real-time PCR System (Applied BiPCaystems, FP Cater City, CA, USA). .. The relative expression levels of microRNA-186-5p and HOXA9 were determined according to the methods described above.

Article Title: Transcriptomic analysis of the prothoracic gland from two lepidopteran insects, domesticated silkmoth Bombyx mori and wild silkmoth Antheraea pernyi
Article Snippet: The total volume of qRT-PCR reactions was 10 µl, containing 3.6 µl of TB Green Premix Ex Taq (TaKaRa), 0.4 µl of specific primers (10 µM), 1 µl of cDNA and 5 µl of ddH2 O. qRT-PCR was performed with a BIO-RAD CFX Connect Real-Time System, and the conditions were as follows: 95 °C for 30 s followed by 40 cycles in 95 °C for 5 s and 60 °C for 30 s. Gene-specific primers used for qRT-PCR analysis are listed in Additional file: Table . .. The mRNA expression levels of the genes of interest were calculated with the 2−ΔΔCt method and nomalized to the abundance of a house-keeping gene, ribosome protein 49 (rp49 ).

Article Title: Quantitative Trait Locus Mapping and Candidate Gene Analysis for Verticillium Wilt Resistance Using Gossypium barbadense Chromosomal Segment Introgressed Line
Article Snippet: Paragraph title: RNA extraction and candidate gene expression analysis qRT-PCR ... All qRT-PCR reactions were performed on an ABI PRISM 7500 Real-Time PCR System using the SYBR Premix Ex Taq II kit (TaKaRa, Japan, Code No. RR820A) according to the manufacturer's instructions.

Article Title: Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma
Article Snippet: GAPDH was used as an endogenous control for the qRT-PCR reactions (TaKaRa, Shiga, Japan). .. The 2−ΔΔCt method was used to calculate the expression of each lncRNA.

Article Title: Heme acts through the Bach1b/Nrf2a-MafK pathway to regulate exocrine peptidase precursor genes in porphyric zebrafish
Article Snippet: RNA extraction and qRT-PCR Total RNA was extracted using the TRIzol® Reagent, according to the manufacturer’s instructions (Invitrogen). cDNA was synthesized by using reverse transcription with the M-MLV reverse transcription kit (Invitrogen), which was then used as the template for qRT-PCR analysis. qRT-PCR reactions were performed with the ABI StepOnePlus™ system, using SYBR® Premix Ex Taq™ (TaKaRa) and the following thermal profile: 95°C for 3 minutes; 95°C for 10 seconds; 58°C for 30 seconds for 40 cycles. .. Relative mRNA expression levels were quantified using the comparative C t (ΔC t ) method and expressed as 2−(ΔΔC t) .

Article Title: A transcriptome study on Macrobrachium nipponense hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)
Article Snippet: Optimized qRT-PCR reactions were performed following the manufacturer’s instructions (SYBR Premix Ex Taq, Takara Bio, Japan.) on a real-time thermal cycler (Bio-Rad, USA), using β-actin as a reference gene. .. Each sample was repeated in triplicate and 2-ΔΔCT methods were used to calculate the expression level of the ten selected genes.

Article Title: GSK3 Inhibitor-BIO Regulates Proliferation of Immortalized Pancreatic Mesenchymal Stem Cells (iPMSCs)
Article Snippet: QRT-PCR The QRT-PCR reactions were set up in 25 µL reaction mixtures containing 12.5 µL 1× SYBR@ PremixExTaqTM (TaKaRa, Biotech. .. The reaction conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec, and 58°C for 20 sec. All expression levels were normalized to β-actin in each well.

Sequencing:

Article Title: A transcriptome study on Macrobrachium nipponense hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)
Article Snippet: The tissues used for qRT-PCR were same as the samples used in transcriptome sequencing. .. Optimized qRT-PCR reactions were performed following the manufacturer’s instructions (SYBR Premix Ex Taq, Takara Bio, Japan.) on a real-time thermal cycler (Bio-Rad, USA), using β-actin as a reference gene.

Concentration Assay:

Article Title: MicroRNA-16 is putatively involved in the NF-κB pathway regulation in ulcerative colitis through adenosine A2a receptor (A2aAR) mRNA targeting
Article Snippet: Real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) For qRT-PCR experiments, total RNA from colonic biopsies or cultured cells were extracted using RNAiso Plus according to the manufacturer’s instructions (Takara, Japan).The concentration and purity of the isolated total RNA samples were verified using an Eppendorf® BioPhotometer Plus (Eppendorf, Hamburg, Germany). .. The One Step PrimeScript® miRNA cDNA Synthesis Kit (Takara, Japan) and PrimeScript® RT Master Mix Perfect Real Time (Takara, Japan) were used to reverse-transcribe the miRNA and mRNA, respectively, according to the manufacturer’s instructions. qRT-PCR reactions were performed in triplicate using SYBR® Premix Ex TaqTM II (Perfect Real Time, Takara, Japan) on LightCycler® (Roche Diagnostics, Nutley, NJ, USA) in a 96-well format, over 45 cycles with denaturation at 95 °C for 10 s and annealing at 60 °C for 20 s. U6 small RNA was used to normalize the levels of miR-16, and GAPDH was used to normalize the mRNA expression levels of A2aAR, IFN-γ, and IL-8.

Software:

Article Title: MicroRNA-186-5p Inhibits Proliferation And Metastasis Of Esophageal Cancer By Mediating HOXA9
Article Snippet: QRT-PCR reactions were performed using SYBR® Premix Ex TaqTM (TaKaRa, Otsu, Japan), and StepOne Plus Real-time PCR System (Applied BiPCaystems, FP Cater City, CA, USA). .. Data analysis was performed using ABI Step One software and the relative expression levels of mRNA were calculated using the 2−ΔΔCt method.

Article Title: A transcriptome study on Macrobrachium nipponense hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)
Article Snippet: Optimized qRT-PCR reactions were performed following the manufacturer’s instructions (SYBR Premix Ex Taq, Takara Bio, Japan.) on a real-time thermal cycler (Bio-Rad, USA), using β-actin as a reference gene. .. Primers for qRT-PCR were carefully designed using Primer Premier 3 software and listed in .

Article Title: Transcriptome analysis of Cucumis sativus infected by Cucurbit chlorotic yellows virus
Article Snippet: Primer sets were designed using Primer Premier software (ver. .. The qRT-PCR reactions were performed with 5 μL of SYBR Green master mix, 20 ng of cDNA, and 200 nM each of the sense and antisense primers, in a total volume of 10 μL (Takara).

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