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    Structured Review

    Thermo Fisher qrt pcr kit
    Discovery of an IFN-inducible subclass of ERVs. ( a ) Immunoblot of pTBK1, TBK1, IKKε, pIRF3, pERK, ERK, pAKT, AKT and tubulin levels in H69 and H69M cells after 24 or 72 h culture. ( b ) Log-2 fold change cytokine/chemokine differences between H69M/H69 CM. ( c ) H E and pTBK1 IHC of a patient-derived SCLC brain metastasis. Scale bar indicates 20 μm. ( d ) Isotype control versus PD-L1 or CD44 surface expression on H69 and H69M cells ± 200 ng/mL 24 h IFNγ stimulation (representative of n=3 biological replicates). ( e ) Immunoblot of pTBK1, TBK1, pERK, ERK, pAKT, AKT and β-actin levels in H69, H69M, H69M-PD-L1 low , and H69M-PD-L1 high cells. ( f ) Log-2 fold change cytokine/chemokines differences between H69M-PD-L1 high or H69M-PD-L1 low /H69 CM. ( g ) <t>qRT-PCR</t> of ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low of previously published ERVs. Error bars are mean ± s.e.m of n=3 biological replicates. TRIM22 promoter and antisense orientation of MLT1C49 in the 3′UTR are represented below the qRT-PCR graph. ( h ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 low cells transfected with pLX-307-GFP control or pLX_307-MLT1C49 construct for 72h. Mean ± s.e.m of n=3 biological replicates shown. ( i ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 high cells transfected with scrambled negative control siRNA or siRNAs specific for MLT1C49. Mean ± s.e.m of n=3 biological replicates shown. ( j ) Overlap of 3′UTR antisense ERVs with H69M upregulated genes (log-2 fold change relative to H69 > 2) and table showing the fold change in expression of these genes/ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low cells. ( k ) Immunoblot of STING, MAVS, pTBK1, TBK1, pIRF3, IRF3, E-Cadherin, Vimentin and β-actin levels in H69M cells after CRISPR mediated deletion of MAVS and/or STING. ( l ) Log-2 fold change cytokine/chemokine differences in CM from H69M cells after CRISPR mediated deletion of MAVS and/or STING compared to sgCTRL (Scramble). ( m ) CXCL10 Luminex absolute levels (pg/mL) in Scramble, STING KO, MAVS KO and double KO (dKO) H69M cells. Mean ± s.e.m of n=2 biological replicates shown. *p
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    Images

    1) Product Images from "Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses"

    Article Title: Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses

    Journal: Nature medicine

    doi: 10.1038/s41591-018-0116-5

    Discovery of an IFN-inducible subclass of ERVs. ( a ) Immunoblot of pTBK1, TBK1, IKKε, pIRF3, pERK, ERK, pAKT, AKT and tubulin levels in H69 and H69M cells after 24 or 72 h culture. ( b ) Log-2 fold change cytokine/chemokine differences between H69M/H69 CM. ( c ) H E and pTBK1 IHC of a patient-derived SCLC brain metastasis. Scale bar indicates 20 μm. ( d ) Isotype control versus PD-L1 or CD44 surface expression on H69 and H69M cells ± 200 ng/mL 24 h IFNγ stimulation (representative of n=3 biological replicates). ( e ) Immunoblot of pTBK1, TBK1, pERK, ERK, pAKT, AKT and β-actin levels in H69, H69M, H69M-PD-L1 low , and H69M-PD-L1 high cells. ( f ) Log-2 fold change cytokine/chemokines differences between H69M-PD-L1 high or H69M-PD-L1 low /H69 CM. ( g ) qRT-PCR of ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low of previously published ERVs. Error bars are mean ± s.e.m of n=3 biological replicates. TRIM22 promoter and antisense orientation of MLT1C49 in the 3′UTR are represented below the qRT-PCR graph. ( h ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 low cells transfected with pLX-307-GFP control or pLX_307-MLT1C49 construct for 72h. Mean ± s.e.m of n=3 biological replicates shown. ( i ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 high cells transfected with scrambled negative control siRNA or siRNAs specific for MLT1C49. Mean ± s.e.m of n=3 biological replicates shown. ( j ) Overlap of 3′UTR antisense ERVs with H69M upregulated genes (log-2 fold change relative to H69 > 2) and table showing the fold change in expression of these genes/ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low cells. ( k ) Immunoblot of STING, MAVS, pTBK1, TBK1, pIRF3, IRF3, E-Cadherin, Vimentin and β-actin levels in H69M cells after CRISPR mediated deletion of MAVS and/or STING. ( l ) Log-2 fold change cytokine/chemokine differences in CM from H69M cells after CRISPR mediated deletion of MAVS and/or STING compared to sgCTRL (Scramble). ( m ) CXCL10 Luminex absolute levels (pg/mL) in Scramble, STING KO, MAVS KO and double KO (dKO) H69M cells. Mean ± s.e.m of n=2 biological replicates shown. *p
    Figure Legend Snippet: Discovery of an IFN-inducible subclass of ERVs. ( a ) Immunoblot of pTBK1, TBK1, IKKε, pIRF3, pERK, ERK, pAKT, AKT and tubulin levels in H69 and H69M cells after 24 or 72 h culture. ( b ) Log-2 fold change cytokine/chemokine differences between H69M/H69 CM. ( c ) H E and pTBK1 IHC of a patient-derived SCLC brain metastasis. Scale bar indicates 20 μm. ( d ) Isotype control versus PD-L1 or CD44 surface expression on H69 and H69M cells ± 200 ng/mL 24 h IFNγ stimulation (representative of n=3 biological replicates). ( e ) Immunoblot of pTBK1, TBK1, pERK, ERK, pAKT, AKT and β-actin levels in H69, H69M, H69M-PD-L1 low , and H69M-PD-L1 high cells. ( f ) Log-2 fold change cytokine/chemokines differences between H69M-PD-L1 high or H69M-PD-L1 low /H69 CM. ( g ) qRT-PCR of ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low of previously published ERVs. Error bars are mean ± s.e.m of n=3 biological replicates. TRIM22 promoter and antisense orientation of MLT1C49 in the 3′UTR are represented below the qRT-PCR graph. ( h ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 low cells transfected with pLX-307-GFP control or pLX_307-MLT1C49 construct for 72h. Mean ± s.e.m of n=3 biological replicates shown. ( i ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 high cells transfected with scrambled negative control siRNA or siRNAs specific for MLT1C49. Mean ± s.e.m of n=3 biological replicates shown. ( j ) Overlap of 3′UTR antisense ERVs with H69M upregulated genes (log-2 fold change relative to H69 > 2) and table showing the fold change in expression of these genes/ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low cells. ( k ) Immunoblot of STING, MAVS, pTBK1, TBK1, pIRF3, IRF3, E-Cadherin, Vimentin and β-actin levels in H69M cells after CRISPR mediated deletion of MAVS and/or STING. ( l ) Log-2 fold change cytokine/chemokine differences in CM from H69M cells after CRISPR mediated deletion of MAVS and/or STING compared to sgCTRL (Scramble). ( m ) CXCL10 Luminex absolute levels (pg/mL) in Scramble, STING KO, MAVS KO and double KO (dKO) H69M cells. Mean ± s.e.m of n=2 biological replicates shown. *p

    Techniques Used: Immunohistochemistry, Derivative Assay, Expressing, Quantitative RT-PCR, Transfection, Construct, Negative Control, CRISPR, Luminex

    Expression of SPARCS-containing genes across cancers. ( a ) ssGSEA of SPARCS-gene containing signature across TCGA (n=3602 tumors) and significantly associated gene sets grouped based on biological annotations. IC = information coefficient. FDR = false discovery rate. ( b ) Intersection of top 1000 genes co-regulated with SPARCS-containing gene signature in TCGA and CCLE datasets. MHC class I pathway genes in top 40 highlighted in red, EMT related genes in blue and immune evasion markers in green. ( c ) Distribution of high versus low SPARCS-containing gene expression by TCGA cancer histology. ( d ) Immunoblot of AXL, MET, Vimentin, STING, MAVS and β-actin levels in cell lines with high, intermediate, or low SPARCS gene signature expression after 72 h culture. ( e ) qRT-PCR of MLT1C49 , CXCL10, PD-L1 and CD44 in SPARCS high and SPARCS low cell lines ± IFNγ 10 min pulse - 24 h chase. p values indicated for comparison of SPARCS high versus SPARCS low groups. Mean ± s.e.m of n=2 biological replicates shown (Two-tailed Mann-Whitney U test). *p
    Figure Legend Snippet: Expression of SPARCS-containing genes across cancers. ( a ) ssGSEA of SPARCS-gene containing signature across TCGA (n=3602 tumors) and significantly associated gene sets grouped based on biological annotations. IC = information coefficient. FDR = false discovery rate. ( b ) Intersection of top 1000 genes co-regulated with SPARCS-containing gene signature in TCGA and CCLE datasets. MHC class I pathway genes in top 40 highlighted in red, EMT related genes in blue and immune evasion markers in green. ( c ) Distribution of high versus low SPARCS-containing gene expression by TCGA cancer histology. ( d ) Immunoblot of AXL, MET, Vimentin, STING, MAVS and β-actin levels in cell lines with high, intermediate, or low SPARCS gene signature expression after 72 h culture. ( e ) qRT-PCR of MLT1C49 , CXCL10, PD-L1 and CD44 in SPARCS high and SPARCS low cell lines ± IFNγ 10 min pulse - 24 h chase. p values indicated for comparison of SPARCS high versus SPARCS low groups. Mean ± s.e.m of n=2 biological replicates shown (Two-tailed Mann-Whitney U test). *p

    Techniques Used: Expressing, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

    2) Product Images from "Astrocytic CCAAT/Enhancer Binding Protein δ Regulates Neuronal Viability and Spatial Learning Ability via miR-135a"

    Article Title: Astrocytic CCAAT/Enhancer Binding Protein δ Regulates Neuronal Viability and Spatial Learning Ability via miR-135a

    Journal: Molecular Neurobiology

    doi: 10.1007/s12035-015-9359-z

    miR-135a suppresses Thbs1/THBS1 transcription through its 3′UTR region. a miR-135a attenuates the expression of Thbs1. Induced miR135a in primary astrocytes with DOX-inducible miR-135a expression system; qRT-PCR and western blot analysis confirmed that miR-135a, Thbs1 mRNAs, and proteins levels, respectively. b Two positions of the Thbs1 3′-untranslated region are predicted to be targets of miR-135a. The seed regions were indicated by the open box (upper panel). Luciferase activity of reporter constructs was measured after co-transfected with pre-miR-135a. c Antisense of miR135a (A-135a) antagonize the effects of Cebpd. Induced A-135a in primary astrocytes with IPTG-inducible A-135a expression system and treated with or without IL-1β treatment. d Induced miR-135a expression repress the THBS1 expression. In stable U373MG cells with DOX-inducible miR-135a expression system, the expression of miR-135a and the level of THBS1 mRNAs and proteins were examined by qRT-PCR, RT-PCR, and Western blot, respectively. e IL-1β and CEBPD suppresses THBS1 transcription via miR-135a binding motif. The seed region was indicated by the open box (upper panel). Luciferase activity of reporter constructs was measured after co-transfected with pre-miR-135a, CEBPD expression vectors, or treated IL-1β. f Antagomir of miR135a (AM135a) dose-dependently antagonized the effects of CEBPD. AM135a or scramble antagomir were transfected into the U373MG stable cells with zinc-inducible CEBPD expression system and then incubated in the presence or absence of 100 μM ZnSO4 for 6 h. RT-PCR and Western blot analysis were performed to examine the expressions of THBS1 mRNA and protein. The data represent the mean ± standard error of three independent experiments, each performed in triplicate. (* P
    Figure Legend Snippet: miR-135a suppresses Thbs1/THBS1 transcription through its 3′UTR region. a miR-135a attenuates the expression of Thbs1. Induced miR135a in primary astrocytes with DOX-inducible miR-135a expression system; qRT-PCR and western blot analysis confirmed that miR-135a, Thbs1 mRNAs, and proteins levels, respectively. b Two positions of the Thbs1 3′-untranslated region are predicted to be targets of miR-135a. The seed regions were indicated by the open box (upper panel). Luciferase activity of reporter constructs was measured after co-transfected with pre-miR-135a. c Antisense of miR135a (A-135a) antagonize the effects of Cebpd. Induced A-135a in primary astrocytes with IPTG-inducible A-135a expression system and treated with or without IL-1β treatment. d Induced miR-135a expression repress the THBS1 expression. In stable U373MG cells with DOX-inducible miR-135a expression system, the expression of miR-135a and the level of THBS1 mRNAs and proteins were examined by qRT-PCR, RT-PCR, and Western blot, respectively. e IL-1β and CEBPD suppresses THBS1 transcription via miR-135a binding motif. The seed region was indicated by the open box (upper panel). Luciferase activity of reporter constructs was measured after co-transfected with pre-miR-135a, CEBPD expression vectors, or treated IL-1β. f Antagomir of miR135a (AM135a) dose-dependently antagonized the effects of CEBPD. AM135a or scramble antagomir were transfected into the U373MG stable cells with zinc-inducible CEBPD expression system and then incubated in the presence or absence of 100 μM ZnSO4 for 6 h. RT-PCR and Western blot analysis were performed to examine the expressions of THBS1 mRNA and protein. The data represent the mean ± standard error of three independent experiments, each performed in triplicate. (* P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Construct, Transfection, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Incubation

    miR-135a is a CEBPD responsive miRNA. a CEBPD induces miR-135a expression. qRT-PCR and Western blot confirmed that miR-135a levels and the expression of HA-tagged CEBPD protein from stable U373MG cells with pMT-CEBPD expression vector, respectively. b CEBPD participate in IL-1β-induced miR-135a expression. qRT-PCR was performed with total RNA harvested from IL-1β-treated U373MG cells with or without attenuation of CEBPD. c miR-135a expression is unaltered in primary Cebpd − / − astrocytes. qRT-PCR was performed using total RNA harvested from primary Cebpd +/+ and Cebpd − / − astrocytes with or without IL-1β treatment. d CEBPD increases miR-135a promoter activities. Schematic representation of reporter constructs with the GLYCTK-AS1-001 promoter. The approximate location of putative CEBPD-binding motif was indicated by open box . A luciferase activity was conducted by co-transfected reporter and expression vectors as indicated in U373MG cells. e CEBPD can directly bind to the GLYCTK-AS1-001 promoter in vivo. A ChIP assay was performed with U373MG cells treated with IL-1β. Chromatin of U373MG cells was separately immunoprecipitated with specific antibody against CEBPD (CD) and control IgG [ 14 ]. The schematic on the top indicated the location of the primers used for detection of the GLYCTK-AS1-001 promoter by PCR. The precipitated DNAs were amplified by PCR with primer (FR-211) on the GLYCTK-AS1-001 promoter (−98/ +113) which contain putative CEBPD-binding motif and negative control primer (FR-250). The data represented the mean ± standard error of three independent experiments, each performed in triplicate. (* P
    Figure Legend Snippet: miR-135a is a CEBPD responsive miRNA. a CEBPD induces miR-135a expression. qRT-PCR and Western blot confirmed that miR-135a levels and the expression of HA-tagged CEBPD protein from stable U373MG cells with pMT-CEBPD expression vector, respectively. b CEBPD participate in IL-1β-induced miR-135a expression. qRT-PCR was performed with total RNA harvested from IL-1β-treated U373MG cells with or without attenuation of CEBPD. c miR-135a expression is unaltered in primary Cebpd − / − astrocytes. qRT-PCR was performed using total RNA harvested from primary Cebpd +/+ and Cebpd − / − astrocytes with or without IL-1β treatment. d CEBPD increases miR-135a promoter activities. Schematic representation of reporter constructs with the GLYCTK-AS1-001 promoter. The approximate location of putative CEBPD-binding motif was indicated by open box . A luciferase activity was conducted by co-transfected reporter and expression vectors as indicated in U373MG cells. e CEBPD can directly bind to the GLYCTK-AS1-001 promoter in vivo. A ChIP assay was performed with U373MG cells treated with IL-1β. Chromatin of U373MG cells was separately immunoprecipitated with specific antibody against CEBPD (CD) and control IgG [ 14 ]. The schematic on the top indicated the location of the primers used for detection of the GLYCTK-AS1-001 promoter by PCR. The precipitated DNAs were amplified by PCR with primer (FR-211) on the GLYCTK-AS1-001 promoter (−98/ +113) which contain putative CEBPD-binding motif and negative control primer (FR-250). The data represented the mean ± standard error of three independent experiments, each performed in triplicate. (* P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Construct, Binding Assay, Luciferase, Activity Assay, Transfection, In Vivo, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Negative Control

    3) Product Images from "MicroRNAs in metamorphic and non-metamorphic transitions in hemimetabolan insect metamorphosis"

    Article Title: MicroRNAs in metamorphic and non-metamorphic transitions in hemimetabolan insect metamorphosis

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-386

    Expression pattern of the 12 miRNA selected from the comparison of the N5 and N6 libraries in Blattella germanica . miRNA levels were measured on nymph 5, nymph 6 and in freshly emerged adults with qRT-PCR; data represent the mean ± SEM, and are indicated as copies of the respective miRNA per 1000 copies of U6; each point represents 3 biological replicates. Schematic patterns of juvenile hormone III (JH) and 20-hydroxyecdysone (20E) titers in nymph 5 and nymph 6 are superimposed in every graphic as empty and grey patterns, respectively. Actual values of JH and 20E titers are represented in the two bottom panels, according to the data of Treiblmayr et al. [ 41 ] (JH) and Romaña et al. [ 15 ] (20E).
    Figure Legend Snippet: Expression pattern of the 12 miRNA selected from the comparison of the N5 and N6 libraries in Blattella germanica . miRNA levels were measured on nymph 5, nymph 6 and in freshly emerged adults with qRT-PCR; data represent the mean ± SEM, and are indicated as copies of the respective miRNA per 1000 copies of U6; each point represents 3 biological replicates. Schematic patterns of juvenile hormone III (JH) and 20-hydroxyecdysone (20E) titers in nymph 5 and nymph 6 are superimposed in every graphic as empty and grey patterns, respectively. Actual values of JH and 20E titers are represented in the two bottom panels, according to the data of Treiblmayr et al. [ 41 ] (JH) and Romaña et al. [ 15 ] (20E).

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of 20-hydroxyecdysone (20E) and 20E plus juvenile hormone III (JH) on miRNA expression in Blattella germanica . Specimens were treated with 1 μg of 20E (A) or with 1 μg of 20E plus 1 μg of JH ( B ) in freshly emerged last instar nymph and miRNAs were measured 24 h later. miRNA levels were measured by qRT-PCR, using U6 as a reference. Data represent 3 biological replicates (mean ± SEM) and are normalized against control females (reference value = 1); the asterisk indicates statistically significant differences with respect to controls (p
    Figure Legend Snippet: Effect of 20-hydroxyecdysone (20E) and 20E plus juvenile hormone III (JH) on miRNA expression in Blattella germanica . Specimens were treated with 1 μg of 20E (A) or with 1 μg of 20E plus 1 μg of JH ( B ) in freshly emerged last instar nymph and miRNAs were measured 24 h later. miRNA levels were measured by qRT-PCR, using U6 as a reference. Data represent 3 biological replicates (mean ± SEM) and are normalized against control females (reference value = 1); the asterisk indicates statistically significant differences with respect to controls (p

    Techniques Used: Expressing, Quantitative RT-PCR

    Effects of miR-252-3p depletion on nymphal development of Blattella germanica . Treated females received two injections of 50 μM of miR-252-3p LNA on day 1 and day 3 of fifth nymphal instar (N5D1 and N5D3, respectively); control females received an equivalent treatment with miRCURY LNA™ microRNA Inhibitor Negative Control A. A ) Levels of miR-252-3p and miR-1-3p (used as negative control) on N5D6. B ) Same data in N5D13. C ) General aspect of a control specimen and a specimen treated with miR-252-3p LNA (LNA-treated) on N5D6. D ) Weight of control and LNA-treated specimens in N5D6. E ) Length (days) of N5 in control and LNA-treated specimens. qRT-PCR data in A and B represent 3 biological replicates and are normalized against the control females (reference value = 1); the asterisk indicates statistically significant differences with respect to controls (p
    Figure Legend Snippet: Effects of miR-252-3p depletion on nymphal development of Blattella germanica . Treated females received two injections of 50 μM of miR-252-3p LNA on day 1 and day 3 of fifth nymphal instar (N5D1 and N5D3, respectively); control females received an equivalent treatment with miRCURY LNA™ microRNA Inhibitor Negative Control A. A ) Levels of miR-252-3p and miR-1-3p (used as negative control) on N5D6. B ) Same data in N5D13. C ) General aspect of a control specimen and a specimen treated with miR-252-3p LNA (LNA-treated) on N5D6. D ) Weight of control and LNA-treated specimens in N5D6. E ) Length (days) of N5 in control and LNA-treated specimens. qRT-PCR data in A and B represent 3 biological replicates and are normalized against the control females (reference value = 1); the asterisk indicates statistically significant differences with respect to controls (p

    Techniques Used: Negative Control, Quantitative RT-PCR

    4) Product Images from "Bacterial Pathogens Activate a Common Inflammatory Pathway through IFN? Regulation of PDCD4"

    Article Title: Bacterial Pathogens Activate a Common Inflammatory Pathway through IFN? Regulation of PDCD4

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003682

    IFNλ regulates PDCD4 in vivo. (A) qRT-PCR analysis of miR-21 in the lungs of WT and IL-28R −/− mice following infection with USA300, µ ± SD. (B) qRT-PCR analysis of PDCD4 mRNA in the lungs of WT and IL-28R −/− mice following infection with USA300, µ ± SD. (C) Western blot analysis of PDCD4 in the lungs of WT and IL-28R −/− mice following infection with USA300, µ ± SD. (D) Numbers (CFU) of USA300 recovered from BAL of WT and PDCD4 −/− mice following 18 hours of infection. (E) Numbers (CFU) of USA300 recovered from lung tissue of WT and PDCD4 −/− mice following 18 hours of infection. (F) Trichrome stained lungs from WT and PDCD4 −/− mice following and 18 hour USA300 infection (original magnification 100x, insert 400x). (G) ELISA analysis of individual cytokines in BAL of WT and PDCD4 −/− mice. Data are representative of at least 2 independent experiments.
    Figure Legend Snippet: IFNλ regulates PDCD4 in vivo. (A) qRT-PCR analysis of miR-21 in the lungs of WT and IL-28R −/− mice following infection with USA300, µ ± SD. (B) qRT-PCR analysis of PDCD4 mRNA in the lungs of WT and IL-28R −/− mice following infection with USA300, µ ± SD. (C) Western blot analysis of PDCD4 in the lungs of WT and IL-28R −/− mice following infection with USA300, µ ± SD. (D) Numbers (CFU) of USA300 recovered from BAL of WT and PDCD4 −/− mice following 18 hours of infection. (E) Numbers (CFU) of USA300 recovered from lung tissue of WT and PDCD4 −/− mice following 18 hours of infection. (F) Trichrome stained lungs from WT and PDCD4 −/− mice following and 18 hour USA300 infection (original magnification 100x, insert 400x). (G) ELISA analysis of individual cytokines in BAL of WT and PDCD4 −/− mice. Data are representative of at least 2 independent experiments.

    Techniques Used: In Vivo, Quantitative RT-PCR, Mouse Assay, Infection, Western Blot, Staining, Enzyme-linked Immunosorbent Assay

    IFNλ regulation of miR-21 and PDCD4. (A) qRT-PCR analysis of IL-8 in 16HBE cells treated with control or PDCD4 siRNA and infected with USA300, µ ± SD. (B) qRT-PCR analysis of miR-21 in 16HBE cells treated with recombinant IFNλ for 1 hour, µ ± SD. (C) Western blot analysis of PDCD4 in 16HBE cells treated with recombinant IFNλ. (D) qRT-PCR analysis of miR-21 in 16HBE cells pretreated with STAT3 inhibitor or DMSO treated with recombinant IFNλ for 1 hour, µ ± SD. (E) Western blot analysis of PDCD4 in 16HBE cells pretreated with STAT3 inhibitor or DMSO treated with recombinant IFNλ for 1 hour. Lysate from 16HBE cells treated with DMSO and IFNλ was used as a positive control (pos). (F) Western blot analysis of PDCD4 in human epithelial cells (16HBE) following stimulation with S. aureus protein A (SpA), µ ± SD. (G) Western blot analysis of PDCD4 in 16HBE cells treated with anti-TNFR1 or control IgG and SPA for 2 hours, µ ± SD. Data are representative of at least 2 independent experiments.
    Figure Legend Snippet: IFNλ regulation of miR-21 and PDCD4. (A) qRT-PCR analysis of IL-8 in 16HBE cells treated with control or PDCD4 siRNA and infected with USA300, µ ± SD. (B) qRT-PCR analysis of miR-21 in 16HBE cells treated with recombinant IFNλ for 1 hour, µ ± SD. (C) Western blot analysis of PDCD4 in 16HBE cells treated with recombinant IFNλ. (D) qRT-PCR analysis of miR-21 in 16HBE cells pretreated with STAT3 inhibitor or DMSO treated with recombinant IFNλ for 1 hour, µ ± SD. (E) Western blot analysis of PDCD4 in 16HBE cells pretreated with STAT3 inhibitor or DMSO treated with recombinant IFNλ for 1 hour. Lysate from 16HBE cells treated with DMSO and IFNλ was used as a positive control (pos). (F) Western blot analysis of PDCD4 in human epithelial cells (16HBE) following stimulation with S. aureus protein A (SpA), µ ± SD. (G) Western blot analysis of PDCD4 in 16HBE cells treated with anti-TNFR1 or control IgG and SPA for 2 hours, µ ± SD. Data are representative of at least 2 independent experiments.

    Techniques Used: Quantitative RT-PCR, Infection, Recombinant, Western Blot, Positive Control

    Induction of IFNλ by S. aureus USA300 inhibits bacterial clearance. (A) qRT-PCR analysis of IFNλ mRNA in BMDCs stimulated with heat killed USA300, µ ± SD. (B) ELISA analysis of IFNλ in BAL of WT mice following 4 and 18 hours of infection with USA300. (C) Numbers (CFU) of USA300 recovered from BAL of WT and IL-28R −/− mice following 4 and 18 hours of infection. (D) Numbers (CFU) of USA300 recovered from lung tissue of WT and IL-28R −/− mice following 4 and 18 hours of infection. (E) Trichrome stained lungs from WT and IL-28R −/− mice following and 18 hour USA300 infection (original magnification 100x, insert 400x). (F) Numbers of dendritic cells (DC), macrophages (MAC), and neutrophils in BAL of WT and IL-28R −/− in unstimulated (NS) mice or following 4 or 18 hours of infection. (G) Numbers of dendritic cells (DC), macrophages (MAC), and neutrophils in lung tissue of WT and IL-28R −/− in unstimulated (NS) mice or following 4 or 18 hours of infection. Data are representative of at least 2 independent experiments.
    Figure Legend Snippet: Induction of IFNλ by S. aureus USA300 inhibits bacterial clearance. (A) qRT-PCR analysis of IFNλ mRNA in BMDCs stimulated with heat killed USA300, µ ± SD. (B) ELISA analysis of IFNλ in BAL of WT mice following 4 and 18 hours of infection with USA300. (C) Numbers (CFU) of USA300 recovered from BAL of WT and IL-28R −/− mice following 4 and 18 hours of infection. (D) Numbers (CFU) of USA300 recovered from lung tissue of WT and IL-28R −/− mice following 4 and 18 hours of infection. (E) Trichrome stained lungs from WT and IL-28R −/− mice following and 18 hour USA300 infection (original magnification 100x, insert 400x). (F) Numbers of dendritic cells (DC), macrophages (MAC), and neutrophils in BAL of WT and IL-28R −/− in unstimulated (NS) mice or following 4 or 18 hours of infection. (G) Numbers of dendritic cells (DC), macrophages (MAC), and neutrophils in lung tissue of WT and IL-28R −/− in unstimulated (NS) mice or following 4 or 18 hours of infection. Data are representative of at least 2 independent experiments.

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

    P. aeruginosa induced cytokine, miR-21, and PDCD4 expression in WT and IL-28R −/− mice. (A) ELISA analysis of individual cytokines in BAL of WT and IL-28R −/− mice. (B) Western blot analysis of MX1 expression in lungs of WT and IL-28R −/− mice following 4 and 18 hours of infection with PAK. (C) qRT-PCR analysis of IL-8 in 16HBE cells treated with control or PDCD4 siRNA and infected with PAK, µ ± SD. (D) qRT-PCR analysis of miR-21 in the lungs of WT and IL-28R −/− mice following infection with PAK, µ ± SD. (E) qRT-PCR analysis of PDCD4 mRNA in the lungs of WT and IL-28R −/− mice following infection with PAK, µ ± SD. (F) Western blot analysis of PDCD4 in the lungs of WT and IL-28R −/− mice following infection with PAK, µ ± SD. (G) Western blot analysis of phosphorylation of p70S6K in 16HBE cells treated with recombinant IFNλ. (H) Western blot analysis of phosphorylation of p70S6K and PDCD4 in the lungs of WT and IL-28R −/− mice following infection with PAK. (I) Western blot analysis of phosphorylation of p70S6K and PDCD4 in the lungs of WT and IL-28R −/− mice following infection with USA300. Data are representative of at least 2 independent experiments.
    Figure Legend Snippet: P. aeruginosa induced cytokine, miR-21, and PDCD4 expression in WT and IL-28R −/− mice. (A) ELISA analysis of individual cytokines in BAL of WT and IL-28R −/− mice. (B) Western blot analysis of MX1 expression in lungs of WT and IL-28R −/− mice following 4 and 18 hours of infection with PAK. (C) qRT-PCR analysis of IL-8 in 16HBE cells treated with control or PDCD4 siRNA and infected with PAK, µ ± SD. (D) qRT-PCR analysis of miR-21 in the lungs of WT and IL-28R −/− mice following infection with PAK, µ ± SD. (E) qRT-PCR analysis of PDCD4 mRNA in the lungs of WT and IL-28R −/− mice following infection with PAK, µ ± SD. (F) Western blot analysis of PDCD4 in the lungs of WT and IL-28R −/− mice following infection with PAK, µ ± SD. (G) Western blot analysis of phosphorylation of p70S6K in 16HBE cells treated with recombinant IFNλ. (H) Western blot analysis of phosphorylation of p70S6K and PDCD4 in the lungs of WT and IL-28R −/− mice following infection with PAK. (I) Western blot analysis of phosphorylation of p70S6K and PDCD4 in the lungs of WT and IL-28R −/− mice following infection with USA300. Data are representative of at least 2 independent experiments.

    Techniques Used: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Infection, Quantitative RT-PCR, Recombinant

    5) Product Images from "Heat Shock Protein 90 (Hsp90) as a Molecular Target for the Development of Novel Drugs Against the Dermatophyte Trichophyton rubrum"

    Article Title: Heat Shock Protein 90 (Hsp90) as a Molecular Target for the Development of Novel Drugs Against the Dermatophyte Trichophyton rubrum

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.01241

    Effect of Hsp90 on the expression of hsp and related genes in T. rubrum . qRT-PCR analyses of the hsp and related genes of T. rubrum grown in malt extract (ME) or keratin medium (KM) in the absence or presence of Hsp90 inhibitor. Gene expression levels are represented by the quantities of mRNA in each condition relative to the control (ME). Data are represented as mean ± SD from three independent experiments with reactions performed in triplicate. Tukey’s ad hoc test was used for statistical analysis ; ∗ P
    Figure Legend Snippet: Effect of Hsp90 on the expression of hsp and related genes in T. rubrum . qRT-PCR analyses of the hsp and related genes of T. rubrum grown in malt extract (ME) or keratin medium (KM) in the absence or presence of Hsp90 inhibitor. Gene expression levels are represented by the quantities of mRNA in each condition relative to the control (ME). Data are represented as mean ± SD from three independent experiments with reactions performed in triplicate. Tukey’s ad hoc test was used for statistical analysis ; ∗ P

    Techniques Used: Expressing, Quantitative RT-PCR

    Transcript levels of T. rubrum hsp genes in response to environmental challenges. (A) qRT-PCR analyses of the transcript levels of hsp genes of T. rubrum grown in malt extract (control), human nail, or human skin fragments for 96 h. (B) Mycelia were transferred to RPMI medium in the absence (control) or presence of sub-inhibitory doses of terbinafine (TRB) or acriflavine (ACR) for 3 h for assessment of drug response. mRNA quantity in each condition relative to the control are represented as mean ± SD from three independent experiments with reactions performed in triplicate. Tukey’s ad hoc test was used for statistical analysis, ∗ P
    Figure Legend Snippet: Transcript levels of T. rubrum hsp genes in response to environmental challenges. (A) qRT-PCR analyses of the transcript levels of hsp genes of T. rubrum grown in malt extract (control), human nail, or human skin fragments for 96 h. (B) Mycelia were transferred to RPMI medium in the absence (control) or presence of sub-inhibitory doses of terbinafine (TRB) or acriflavine (ACR) for 3 h for assessment of drug response. mRNA quantity in each condition relative to the control are represented as mean ± SD from three independent experiments with reactions performed in triplicate. Tukey’s ad hoc test was used for statistical analysis, ∗ P

    Techniques Used: Quantitative RT-PCR

    6) Product Images from "Novel Tissue-Specific Mechanism of Regulation of Angiogenesis and Cancer Growth in Response to Hyperglycemia"

    Article Title: Novel Tissue-Specific Mechanism of Regulation of Angiogenesis and Cancer Growth in Response to Hyperglycemia

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.112.005967

    Prostate cancer growth in hyperglycemic mice. RM1 cancer cells were injected subcutaneously to hyper‐ or normoglycemic mice as described in Methods. Tumors excised a week later were weighed (P1, n=10 in control group, n=11 in hyperglycemic group), stained with anti‐TSP‐1 and anti‐CD31 antibodies, and TSP‐1 mRNA and miR‐467 levels were measured by QRT‐PCR. Levels of proteins and RNA for each individual mouse are shown on the graph. In P2, P3, P5, and P6, n=6 to 10 because of an insufficient amount of tissue to accomplish all the analyses in some samples. For the analyses of immunohistochemical staining (P2 and P6), 5 to 11 fields from each plug were quantified, depending on the size of the tumor (total number of fields > 85 for each protein in either the diabetic or the normoglycemic mice). In P4, data from both the normoglycemic and hyperglycemic groups are combined, and n=15. NS indicates difference between means is not statistically significant; TSP, thrombospondin; RT‐PCR, reverse‐transcription polymerase chain reaction.
    Figure Legend Snippet: Prostate cancer growth in hyperglycemic mice. RM1 cancer cells were injected subcutaneously to hyper‐ or normoglycemic mice as described in Methods. Tumors excised a week later were weighed (P1, n=10 in control group, n=11 in hyperglycemic group), stained with anti‐TSP‐1 and anti‐CD31 antibodies, and TSP‐1 mRNA and miR‐467 levels were measured by QRT‐PCR. Levels of proteins and RNA for each individual mouse are shown on the graph. In P2, P3, P5, and P6, n=6 to 10 because of an insufficient amount of tissue to accomplish all the analyses in some samples. For the analyses of immunohistochemical staining (P2 and P6), 5 to 11 fields from each plug were quantified, depending on the size of the tumor (total number of fields > 85 for each protein in either the diabetic or the normoglycemic mice). In P4, data from both the normoglycemic and hyperglycemic groups are combined, and n=15. NS indicates difference between means is not statistically significant; TSP, thrombospondin; RT‐PCR, reverse‐transcription polymerase chain reaction.

    Techniques Used: Mouse Assay, Injection, Staining, Quantitative RT-PCR, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction

    miR‐467 regulates breast cancer growth in STZ‐treated hyperglycemic mice. EMT6 cancer cells were injected subcutaneously into STZ‐treated hyperglycemic or normoglycemic mice as described in Methods. Tumors excised a week later were weighed (B1), stained with anti‐TSP‐1 (B2), anti‐CD31 (B7), and α‐actin antibodies (B8). TSP‐1 mRNA (B3) and miR‐467 (B5) levels were measured by QRT‐PCR. Levels of proteins and RNA for each individual mouse are shown on the graph. A, STZ‐induced hyperglycemia, n=10 in control group and n=16 in hyperglycemic group. In B4, B6, and B7, data from both the normoglycemic and hyperglycemic groups were combined, n=25. For the analyses of immunohistological staining (B2 and B8), large field‐of‐view (FOV) images were generated (see Methods for details) from each tumor sections. * P
    Figure Legend Snippet: miR‐467 regulates breast cancer growth in STZ‐treated hyperglycemic mice. EMT6 cancer cells were injected subcutaneously into STZ‐treated hyperglycemic or normoglycemic mice as described in Methods. Tumors excised a week later were weighed (B1), stained with anti‐TSP‐1 (B2), anti‐CD31 (B7), and α‐actin antibodies (B8). TSP‐1 mRNA (B3) and miR‐467 (B5) levels were measured by QRT‐PCR. Levels of proteins and RNA for each individual mouse are shown on the graph. A, STZ‐induced hyperglycemia, n=10 in control group and n=16 in hyperglycemic group. In B4, B6, and B7, data from both the normoglycemic and hyperglycemic groups were combined, n=25. For the analyses of immunohistological staining (B2 and B8), large field‐of‐view (FOV) images were generated (see Methods for details) from each tumor sections. * P

    Techniques Used: Mouse Assay, Injection, Staining, Quantitative RT-PCR, Generated

    7) Product Images from "Highly Sensitive Quantitative Real-Time PCR for the Detection of Plasmodium Liver-Stage Parasite Burden following Low-Dose Sporozoite Challenge"

    Article Title: Highly Sensitive Quantitative Real-Time PCR for the Detection of Plasmodium Liver-Stage Parasite Burden following Low-Dose Sporozoite Challenge

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077811

    Standard Curves. qRT-PCR analysis of a dilution series ranging from 10 7 to 1 copies of Py18S (A) and PyCytB (B) control plasmid. Data are presented as mean values for three independent runs with two technical replicates each. Error bars represent mean standard deviation between technical replicates of each run.
    Figure Legend Snippet: Standard Curves. qRT-PCR analysis of a dilution series ranging from 10 7 to 1 copies of Py18S (A) and PyCytB (B) control plasmid. Data are presented as mean values for three independent runs with two technical replicates each. Error bars represent mean standard deviation between technical replicates of each run.

    Techniques Used: Quantitative RT-PCR, Plasmid Preparation, Standard Deviation

    Limit of Detection after infection with different numbers of cryopreserved sporozoites. Detection of (A) Py18S rRNA or (B) PyCytB mRNA in liver RNA extracts of BALB/c mice 40h after infection with 50, 100, 500, 1000, 1500, 2000 or 5000 cryopreserved sporozoites. Data are represented as mean ‘plasmid equivalent’ for each group (n=3-7 mice/group). Two technical replicates of each mouse sample were run twice in independent qRT-PCR experiments and the mean ‘plasmid equivalent’ measured for each mouse/group was calculated. The error bars represent the mean variation between mice receiving the same sporozoite dose, calculated as the standard error for each group. A linear trendline was fitted to the data points to represent the correlation of measured values to the number of injected sporozoites. The dotted line represents the limit of detection defined as the mean value of target cDNA per 10 6 copies GAPDH within the group of samples for which a maximum of 5% of reactions failed. Statistical significance between groups of mice receiving different sporozoite doses was evaluated using One-way ANOVA followed by two-tailed Mann-Whitney test, * p
    Figure Legend Snippet: Limit of Detection after infection with different numbers of cryopreserved sporozoites. Detection of (A) Py18S rRNA or (B) PyCytB mRNA in liver RNA extracts of BALB/c mice 40h after infection with 50, 100, 500, 1000, 1500, 2000 or 5000 cryopreserved sporozoites. Data are represented as mean ‘plasmid equivalent’ for each group (n=3-7 mice/group). Two technical replicates of each mouse sample were run twice in independent qRT-PCR experiments and the mean ‘plasmid equivalent’ measured for each mouse/group was calculated. The error bars represent the mean variation between mice receiving the same sporozoite dose, calculated as the standard error for each group. A linear trendline was fitted to the data points to represent the correlation of measured values to the number of injected sporozoites. The dotted line represents the limit of detection defined as the mean value of target cDNA per 10 6 copies GAPDH within the group of samples for which a maximum of 5% of reactions failed. Statistical significance between groups of mice receiving different sporozoite doses was evaluated using One-way ANOVA followed by two-tailed Mann-Whitney test, * p

    Techniques Used: Infection, Mouse Assay, Plasmid Preparation, Quantitative RT-PCR, Injection, Two Tailed Test, MANN-WHITNEY

    Accuracy of parasite cDNA detection. The standard deviation for four replicates of one sample was divided by the mean value for the target gene and plotted against the log of the mean parasite-derived cDNA copies/reaction. Each point represents the mean of four replicates of one mouse liver cDNA sample. A cut-off value of 8x10 -3 for the ratio between standard deviation and mean is suggested for accurate quantitation. The dotted line marks the mean value between the highest amount of parasite cDNA measured with a standard deviation/mean ratio below 8x10 -3 and the highest amount of parasite cDNA measured with a standard deviation/mean ratio above 8x10 -3 , for (A) Py18S qRT-PCR (1.6 copies/reaction) and for (B) PyCytB qRT-PCR (66.5 copies/reaction).
    Figure Legend Snippet: Accuracy of parasite cDNA detection. The standard deviation for four replicates of one sample was divided by the mean value for the target gene and plotted against the log of the mean parasite-derived cDNA copies/reaction. Each point represents the mean of four replicates of one mouse liver cDNA sample. A cut-off value of 8x10 -3 for the ratio between standard deviation and mean is suggested for accurate quantitation. The dotted line marks the mean value between the highest amount of parasite cDNA measured with a standard deviation/mean ratio below 8x10 -3 and the highest amount of parasite cDNA measured with a standard deviation/mean ratio above 8x10 -3 , for (A) Py18S qRT-PCR (1.6 copies/reaction) and for (B) PyCytB qRT-PCR (66.5 copies/reaction).

    Techniques Used: Standard Deviation, Derivative Assay, Quantitation Assay, Quantitative RT-PCR

    8) Product Images from "CCR7 promote lymph node metastasis via regulating VEGF-C/D-R3 pathway in lung adenocarcinoma"

    Article Title: CCR7 promote lymph node metastasis via regulating VEGF-C/D-R3 pathway in lung adenocarcinoma

    Journal: Journal of Cancer

    doi: 10.7150/jca.19069

    The effect of CCR7 on tumorigenesis in vivo. (A) CCR7-siRNA lentivector was transfected into A549 cells, which were injected in male athymic mice, respectively. (B) Tumor weights were calculated after injection 8 weeks. Bars indicate S.D. (C) qRT-PCR was performed to detect the expression of CCR7, VEGF-C, VEGF-D, VEGF-R3 and LYVE-1 in tumor nodule. β-actin was used as an internal control. * P
    Figure Legend Snippet: The effect of CCR7 on tumorigenesis in vivo. (A) CCR7-siRNA lentivector was transfected into A549 cells, which were injected in male athymic mice, respectively. (B) Tumor weights were calculated after injection 8 weeks. Bars indicate S.D. (C) qRT-PCR was performed to detect the expression of CCR7, VEGF-C, VEGF-D, VEGF-R3 and LYVE-1 in tumor nodule. β-actin was used as an internal control. * P

    Techniques Used: In Vivo, Transfection, Injection, Mouse Assay, Quantitative RT-PCR, Expressing

    9) Product Images from "Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells"

    Article Title: Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells

    Journal: RNA

    doi: 10.1261/rna.045559.114

    Northern blots and qRT-PCR
    Figure Legend Snippet: Northern blots and qRT-PCR

    Techniques Used: Northern Blot, Quantitative RT-PCR

    10) Product Images from "Aedes aegypti microRNA miR-2b regulates ubiquitin-related modifier to control chikungunya virus replication"

    Article Title: Aedes aegypti microRNA miR-2b regulates ubiquitin-related modifier to control chikungunya virus replication

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18043-0

    qRT-PCR analysis of selected six miRNAs showing differential regulation upon CHIKV infection at 12, 24, and 48 h, validating deep sequencing results. Data were expressed as mean ± SEM; **** p
    Figure Legend Snippet: qRT-PCR analysis of selected six miRNAs showing differential regulation upon CHIKV infection at 12, 24, and 48 h, validating deep sequencing results. Data were expressed as mean ± SEM; **** p

    Techniques Used: Quantitative RT-PCR, Infection, Sequencing

    11) Product Images from "miR-10a is aberrantly overexpressed in Nucleophosmin1 mutated acute myeloid leukaemia and its suppression induces cell death"

    Article Title: miR-10a is aberrantly overexpressed in Nucleophosmin1 mutated acute myeloid leukaemia and its suppression induces cell death

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-11-8

    Unique microRNA signature of NPM1 mut AML is characterised by miR-10a over-expression . A Hierarchical cluster analysis of 28 AML samples according to NPM1 mutational status, with over-expression of miR-10a, let-7b, let-7c and under-expression of miR-130a and miR-335, with accompanying fold change B . C Validation of miR-10a expression by qRT-PCR in normal bone marrow, NPM1 wt - AML and NPM1 mut -AML respectively, with values presented normalised to the mean of the normal bone marrow samples. D miR-10a expression by qRT-PCR in selected malignant cell lines: MV4-11, OCI-AML3 (myelomonoblastic) and THP-1 (monoblastic). The expression values are depicted relative to that of MV4-11 cells, which had the lowest miR-10a expression. Error bars denote SEM. * p
    Figure Legend Snippet: Unique microRNA signature of NPM1 mut AML is characterised by miR-10a over-expression . A Hierarchical cluster analysis of 28 AML samples according to NPM1 mutational status, with over-expression of miR-10a, let-7b, let-7c and under-expression of miR-130a and miR-335, with accompanying fold change B . C Validation of miR-10a expression by qRT-PCR in normal bone marrow, NPM1 wt - AML and NPM1 mut -AML respectively, with values presented normalised to the mean of the normal bone marrow samples. D miR-10a expression by qRT-PCR in selected malignant cell lines: MV4-11, OCI-AML3 (myelomonoblastic) and THP-1 (monoblastic). The expression values are depicted relative to that of MV4-11 cells, which had the lowest miR-10a expression. Error bars denote SEM. * p

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

    12) Product Images from "Affinity Purification of Binding miRNAs for Messenger RNA Fused with a Common Tag"

    Article Title: Affinity Purification of Binding miRNAs for Messenger RNA Fused with a Common Tag

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms150814753

    mRNA:miRNAs isolation technique for Eps8 and its target miRNAs. ( A ) The enrichment of RNA chimeric mRNAs of EGFP -3'-UTR demonstrated the efficiency of isolation technique; ( B ) The regulated miRNAs of Eps8 were obtained from the product that was transfected with 3'-UTR-Eps8 through semi-quantitative PCR. At the same time, miRNAs that without binding sites could not be amplified from these products; ( C ) The levels of detected miRNAs were verified using qRT-PCR after the process of isolation technique; and ( D ) qRT-PCR showing the relationship between mRNA and their target miRNA after the isolation. RT: Reverse Transcriptase.
    Figure Legend Snippet: mRNA:miRNAs isolation technique for Eps8 and its target miRNAs. ( A ) The enrichment of RNA chimeric mRNAs of EGFP -3'-UTR demonstrated the efficiency of isolation technique; ( B ) The regulated miRNAs of Eps8 were obtained from the product that was transfected with 3'-UTR-Eps8 through semi-quantitative PCR. At the same time, miRNAs that without binding sites could not be amplified from these products; ( C ) The levels of detected miRNAs were verified using qRT-PCR after the process of isolation technique; and ( D ) qRT-PCR showing the relationship between mRNA and their target miRNA after the isolation. RT: Reverse Transcriptase.

    Techniques Used: Isolation, Transfection, Real-time Polymerase Chain Reaction, Binding Assay, Amplification, Quantitative RT-PCR

    Validation of miRNAs targeting to single mRNA isolation technique. The RNA in the products of pull-down were reverse transcripted and as templates for amplifying. The chimeric mRNAs of EGFP -3'-UTR were detected from corresponding products of pull-down ( A ); The enrichment of miRNAs targeting to 3'-UTR- PDCD4 were shown in the pull-down products using semi-quantitative PCR ( B ) and qRT-PCR ( C ); The 3'-UTRs and miR-21 levels were detected from the pull-down products ( D ). RT: Reverse Transcriptase.
    Figure Legend Snippet: Validation of miRNAs targeting to single mRNA isolation technique. The RNA in the products of pull-down were reverse transcripted and as templates for amplifying. The chimeric mRNAs of EGFP -3'-UTR were detected from corresponding products of pull-down ( A ); The enrichment of miRNAs targeting to 3'-UTR- PDCD4 were shown in the pull-down products using semi-quantitative PCR ( B ) and qRT-PCR ( C ); The 3'-UTRs and miR-21 levels were detected from the pull-down products ( D ). RT: Reverse Transcriptase.

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

    13) Product Images from "Research Resource: The Dexamethasone Transcriptome in Hypothalamic Embryonic Neural Stem Cells"

    Article Title: Research Resource: The Dexamethasone Transcriptome in Hypothalamic Embryonic Neural Stem Cells

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2015-1258

    qRT-PCR validations of sex-specific GR-target genes in hypothalamic NPSCs. Heat map depicting significant GR-target genes (A), quantification of GR-target genes similarly and differentially in male and female hypothalamic NPSCs (B), qRT-PCR validation
    Figure Legend Snippet: qRT-PCR validations of sex-specific GR-target genes in hypothalamic NPSCs. Heat map depicting significant GR-target genes (A), quantification of GR-target genes similarly and differentially in male and female hypothalamic NPSCs (B), qRT-PCR validation

    Techniques Used: Quantitative RT-PCR

    Boxplots of novel GR-target gene HIF3 α identified by RNA-Seq analysis (A) and qRT-PCR validations in both male and female hypothalamic NPSCs in vitro at 4 (B), 8 (C), and 24 (D) hours and within the hypothalamus at E15.5 after 24 hours of dex
    Figure Legend Snippet: Boxplots of novel GR-target gene HIF3 α identified by RNA-Seq analysis (A) and qRT-PCR validations in both male and female hypothalamic NPSCs in vitro at 4 (B), 8 (C), and 24 (D) hours and within the hypothalamus at E15.5 after 24 hours of dex

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR, In Vitro

    14) Product Images from "Non-Coding RNAs are Differentially Expressed by Nocardia brasiliensis in Vitro and in Experimental Actinomycetoma"

    Article Title: Non-Coding RNAs are Differentially Expressed by Nocardia brasiliensis in Vitro and in Experimental Actinomycetoma

    Journal: The Open Microbiology Journal

    doi: 10.2174/1874285801711010112

    The validation of the RNA-Seq results by qRT-PCR and changes in the in vitro and in vivo expression levels. Differential expression levels of eight selected transcripts were determined. The Y-axis values indicate the fold change differences in expression and were calculated using the 2 ΔΔCt parameter. qRT-PCR data represent the mean of three independent experiments. Error bars represent the standard deviation.
    Figure Legend Snippet: The validation of the RNA-Seq results by qRT-PCR and changes in the in vitro and in vivo expression levels. Differential expression levels of eight selected transcripts were determined. The Y-axis values indicate the fold change differences in expression and were calculated using the 2 ΔΔCt parameter. qRT-PCR data represent the mean of three independent experiments. Error bars represent the standard deviation.

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR, In Vitro, In Vivo, Expressing, Standard Deviation

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    Article Snippet: .. Forty cycles were set for qRT-PCR program and the abundances of interested gene expression were calculated following the formula (2 -ΔΔCt ) suggested in the instruction of qRT-PCR kit (Applied Biosystems). .. Specific primers used for the qRT-PCR analysis are as follows: for GAPDH , 5′-CCACCCAGAAGACTGTGGAT-3′ and 5′-AAGGTCATCCCTGAGCTGAA-3′; for THBS1 , 5′-GACCTGCCACATTCAGGAGT-3′ and 5′-CTTTCTTGCAGGCTTTGGTC-3′; for Thbs1 , 5′-CCAAAGCCTGCAAGAAAGAC-3′ and 5′-CCTGCTTGTTGCAAACTTGA-3′; for miR-135a reverse transcription (RT) primer, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATCACTTGAGTCACATAG-3′; and for miR-135a forward, 5′-ACACTCCAGCTCAGTATGGCTTTTTATTCCTATGT-3′ and reverse, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAG-3′.

    Article Title: Aedes aegypti microRNA miR-2b regulates ubiquitin-related modifier to control chikungunya virus replication
    Article Snippet: .. For miRNAs, 1 µg of RNA was polyadenylated, reverse transcribed, and quantified by qRT-PCR using NCode miRNA First Strand cDNA Synthesis and qRT-PCR kit (Thermo Fisher Scientific, MA). qRT-PCR reactions were set up using 1:10 diluted cDNA as template following manufacturer instructions. .. For other transcripts, cDNA was used directly for quantitative RT-PCR using QuantiTect SYBR Green RT-PCR Kit (Qiagen, Germany).

    Article Title: Non-Coding RNAs are Differentially Expressed by Nocardia brasiliensis in Vitro and in Experimental Actinomycetoma
    Article Snippet: .. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) To confirm the presence of the transcripts found by RNA-Seq, qRT-PCR was performed using the NCode™ miRNA First-Strand cDNA Synthesis and qRT-PCR kit (Life Technologies Corporation, cat. no. MIRQ-100) according to the manufacturer´s instructions. .. As starting material we used 1 µg RNA obtained from infection or in vitro growth conditions.

    Article Title: CCR7 promote lymph node metastasis via regulating VEGF-C/D-R3 pathway in lung adenocarcinoma
    Article Snippet: .. The qRT-PCR kit was provided by Invitrogen. ..

    Article Title: Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses
    Article Snippet: .. After extraction, 1 μg total RNA was used to generate cDNA with the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR kit, which includes both oligo-dT and random primers (Thermo Fisher Scientific, Waltham, MA). .. Quantitative reverse transcription PCR (qRT-PCR) of the indicated genes ( ) was performed using SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA) and the Applied Biosystems 7300 Fast real-time PCR system and software.

    Article Title: Novel Tissue-Specific Mechanism of Regulation of Angiogenesis and Cancer Growth in Response to Hyperglycemia
    Article Snippet: .. To measure miRNA levels, 1 μg of total RNA was first polyadenylated followed by first‐strand cDNA synthesis using the protocol for NCode miRNA First‐Strand cDNA Synthesis and a qRT‐PCR kit (Invitrogen). .. Real‐time PCR amplification was performed with reagents from the same kit.

    Article Title: Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells
    Article Snippet: .. The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen). .. For validation of small RNAs, one universal qPCR reverse primer was used to amplify the poly-dT region of the cDNA along with small RNA specific forward primers ( ).

    Article Title: Research Resource: The Dexamethasone Transcriptome in Hypothalamic Embryonic Neural Stem Cells
    Article Snippet: .. The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen) and one universal qPCR reverse primer was used to amplify the poly-dT region of the cDNA T1 along with small RNA-specific forward primers (3′ or 5′). qRT-PCRs were performed on a Bio-Rad CFX qRT-PCR machine using iTaq Universal SYBR Green Supermix (172-5121; Bio-Rad) and primers with efficiencies calculated to be greater than 80% ( ). .. There was high-level expression ( > 330 FPKMs) and no differences observed due to sex or treatment for glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) in our RNA-Seq dataset, and therefore, relative expression was calculated with Gapdh as the reference gene, whereas for microRNAs, U6 served as the reference gene.

    Article Title: Affinity Purification of Binding miRNAs for Messenger RNA Fused with a Common Tag
    Article Snippet: .. The reverse qRT-PCR primers of the miRNAs were used NCode™ miRNA First-Strand cDNA Synthesis and qRT-PCR kit (Invitrogen, Carlsbad, CA, USA). .. Statical Analysis GraphPad Prism 5 was used for statistical analysis.

    Article Title: miR-10a is aberrantly overexpressed in Nucleophosmin1 mutated acute myeloid leukaemia and its suppression induces cell death
    Article Snippet: .. To confirm miR-10a expression in primary samples and comparison of expression in cell lines, 1 μg of total RNA was used for cDNA synthesis reaction using the NCode microRNA first strand synthesis and qRT-PCR kit (Invitrogen, USA). .. Quantitative real time polymerase chain reaction (qRT-PCR) was performed using the Platinum Sybr Green Taq (Invitrogen, USA) and the Rotorgene RG-3000 thermocycler (Qiagen, Hilden, Germany).

    SYBR Green Assay:

    Article Title: Bacterial Pathogens Activate a Common Inflammatory Pathway through IFN? Regulation of PDCD4
    Article Snippet: Quantitative real-time RT-PCR (QRT-PCR) was performed using Power SYBR Green PCR Master Mix in a Step One Plus Thermal Cycler (Applied Biosystems). .. For miRNA analysis cDNA was generated and qRT-PCR was run using NCode miRNA First-Strand cDNA Synthesis and qRT-PCR Kit (Life Technologies) according to the manufacturers instructions.

    Article Title: MicroRNAs in metamorphic and non-metamorphic transitions in hemimetabolan insect metamorphosis
    Article Snippet: An amount of 400 ng of total RNA was reverse transcribed with the NCode miRNA first-strand synthesis and qRT-PCR kit (Invitrogen), following the manufacturer protocol. .. Amplification reactions were carried out using IQTM SYBR Green Supermix (BioRad) and the following protocol: 95°C for 2 min, and 40 cycles of 95°C for 15 s and 60°C for 30 s, in a MyIQ Real-Time PCR Detection System (BioRad).

    Article Title: Aedes aegypti microRNA miR-2b regulates ubiquitin-related modifier to control chikungunya virus replication
    Article Snippet: For miRNAs, 1 µg of RNA was polyadenylated, reverse transcribed, and quantified by qRT-PCR using NCode miRNA First Strand cDNA Synthesis and qRT-PCR kit (Thermo Fisher Scientific, MA). qRT-PCR reactions were set up using 1:10 diluted cDNA as template following manufacturer instructions. .. For other transcripts, cDNA was used directly for quantitative RT-PCR using QuantiTect SYBR Green RT-PCR Kit (Qiagen, Germany).

    Article Title: Salt Stress Effects on Secondary Metabolites of Cotton in Relation to Gene Expression Responsible for Aphid Development
    Article Snippet: .. The first-strand cDNA was synthesized from 1μg of total RNA with First-Strand Synthesis SuperMix for qRT-PCR kit (Invitrogen). qRT-PCR reactions were performed using the iCycler iQ2 sequence detection system (Bio-Rad, Hercules, CA) with SYBR Green PCR Master Mix (Applied Biosystems). .. The PCR parameters were 95°C for 3min; 40 cycles at 95°C for 10s, at annealing temperatures of 60°C for 10s, and 72°C for 10s.

    Article Title: Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses
    Article Snippet: After extraction, 1 μg total RNA was used to generate cDNA with the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR kit, which includes both oligo-dT and random primers (Thermo Fisher Scientific, Waltham, MA). .. Quantitative reverse transcription PCR (qRT-PCR) of the indicated genes ( ) was performed using SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA) and the Applied Biosystems 7300 Fast real-time PCR system and software.

    Article Title: Research Resource: The Dexamethasone Transcriptome in Hypothalamic Embryonic Neural Stem Cells
    Article Snippet: .. The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen) and one universal qPCR reverse primer was used to amplify the poly-dT region of the cDNA T1 along with small RNA-specific forward primers (3′ or 5′). qRT-PCRs were performed on a Bio-Rad CFX qRT-PCR machine using iTaq Universal SYBR Green Supermix (172-5121; Bio-Rad) and primers with efficiencies calculated to be greater than 80% ( ). .. There was high-level expression ( > 330 FPKMs) and no differences observed due to sex or treatment for glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) in our RNA-Seq dataset, and therefore, relative expression was calculated with Gapdh as the reference gene, whereas for microRNAs, U6 served as the reference gene.

    Article Title: Affinity Purification of Binding miRNAs for Messenger RNA Fused with a Common Tag
    Article Snippet: The resulting cDNA was amplified using oligonucleotide primers and SYBR Green MasterMix (TaKaRa, Dalian, China) for qRT-PCR. .. The reverse qRT-PCR primers of the miRNAs were used NCode™ miRNA First-Strand cDNA Synthesis and qRT-PCR kit (Invitrogen, Carlsbad, CA, USA).

    Article Title: miR-10a is aberrantly overexpressed in Nucleophosmin1 mutated acute myeloid leukaemia and its suppression induces cell death
    Article Snippet: To confirm miR-10a expression in primary samples and comparison of expression in cell lines, 1 μg of total RNA was used for cDNA synthesis reaction using the NCode microRNA first strand synthesis and qRT-PCR kit (Invitrogen, USA). .. Quantitative real time polymerase chain reaction (qRT-PCR) was performed using the Platinum Sybr Green Taq (Invitrogen, USA) and the Rotorgene RG-3000 thermocycler (Qiagen, Hilden, Germany).

    Microarray:

    Article Title: miR-10a is aberrantly overexpressed in Nucleophosmin1 mutated acute myeloid leukaemia and its suppression induces cell death
    Article Snippet: Paragraph title: microRNA microarray and qRT-PCR ... To confirm miR-10a expression in primary samples and comparison of expression in cell lines, 1 μg of total RNA was used for cDNA synthesis reaction using the NCode microRNA first strand synthesis and qRT-PCR kit (Invitrogen, USA).

    Incubation:

    Article Title: Non-Coding RNAs are Differentially Expressed by Nocardia brasiliensis in Vitro and in Experimental Actinomycetoma
    Article Snippet: Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) To confirm the presence of the transcripts found by RNA-Seq, qRT-PCR was performed using the NCode™ miRNA First-Strand cDNA Synthesis and qRT-PCR kit (Life Technologies Corporation, cat. no. MIRQ-100) according to the manufacturer´s instructions. .. Briefly, a poly A tail was added to RNA of sizes < 200 nt using the poly A polymerase and incubated at 37°C for 15 min.

    Luciferase:

    Article Title: Novel Tissue-Specific Mechanism of Regulation of Angiogenesis and Cancer Growth in Response to Hyperglycemia
    Article Snippet: The conditions and primers used to measure TSP‐1 and luciferase mRNA levels were described previously. .. To measure miRNA levels, 1 μg of total RNA was first polyadenylated followed by first‐strand cDNA synthesis using the protocol for NCode miRNA First‐Strand cDNA Synthesis and a qRT‐PCR kit (Invitrogen).

    Expressing:

    Article Title: Highly Sensitive Quantitative Real-Time PCR for the Detection of Plasmodium Liver-Stage Parasite Burden following Low-Dose Sporozoite Challenge
    Article Snippet: .. A commercially available qRT-PCR kit (TaqMan® Gene Expression Assays from Applied Biosystems, Life Technologies Australia Pty Ltd., Mulgrave, VIC) was used to amplify mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene for the normalisation of Py18S or PyCytB values. .. 6. qRT-PCR Py18S and PyCytB qRT-PCR conditions were determined empirically by testing a range of primer concentrations (range 0.2µM-1µM) and probe concentrations (range 0.1µM-0.5µM) with different PCR reaction mixes in 15µl reactions with 2µl cDNA.

    Article Title: Heat Shock Protein 90 (Hsp90) as a Molecular Target for the Development of Novel Drugs Against the Dermatophyte Trichophyton rubrum
    Article Snippet: Paragraph title: Gene Expression Analysis ... First-strand cDNA was synthesized using the SuperScriptIII First-Strand Synthesis Super Mix for qRT-PCR kit (Invitrogen).

    Article Title: Bacterial Pathogens Activate a Common Inflammatory Pathway through IFN? Regulation of PDCD4
    Article Snippet: IL-8 and PDCD4 expression was normalized to actin. .. For miRNA analysis cDNA was generated and qRT-PCR was run using NCode miRNA First-Strand cDNA Synthesis and qRT-PCR Kit (Life Technologies) according to the manufacturers instructions.

    Article Title: MicroRNAs in metamorphic and non-metamorphic transitions in hemimetabolan insect metamorphosis
    Article Snippet: Paragraph title: Expression patterns ... An amount of 400 ng of total RNA was reverse transcribed with the NCode miRNA first-strand synthesis and qRT-PCR kit (Invitrogen), following the manufacturer protocol.

    Article Title: Astrocytic CCAAT/Enhancer Binding Protein δ Regulates Neuronal Viability and Spatial Learning Ability via miR-135a
    Article Snippet: .. Forty cycles were set for qRT-PCR program and the abundances of interested gene expression were calculated following the formula (2 -ΔΔCt ) suggested in the instruction of qRT-PCR kit (Applied Biosystems). .. Specific primers used for the qRT-PCR analysis are as follows: for GAPDH , 5′-CCACCCAGAAGACTGTGGAT-3′ and 5′-AAGGTCATCCCTGAGCTGAA-3′; for THBS1 , 5′-GACCTGCCACATTCAGGAGT-3′ and 5′-CTTTCTTGCAGGCTTTGGTC-3′; for Thbs1 , 5′-CCAAAGCCTGCAAGAAAGAC-3′ and 5′-CCTGCTTGTTGCAAACTTGA-3′; for miR-135a reverse transcription (RT) primer, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATCACTTGAGTCACATAG-3′; and for miR-135a forward, 5′-ACACTCCAGCTCAGTATGGCTTTTTATTCCTATGT-3′ and reverse, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAG-3′.

    Article Title: Aedes aegypti microRNA miR-2b regulates ubiquitin-related modifier to control chikungunya virus replication
    Article Snippet: Quantitative RT-PCR Expression profiling was carried out by quantitative RT-PCR. .. For miRNAs, 1 µg of RNA was polyadenylated, reverse transcribed, and quantified by qRT-PCR using NCode miRNA First Strand cDNA Synthesis and qRT-PCR kit (Thermo Fisher Scientific, MA). qRT-PCR reactions were set up using 1:10 diluted cDNA as template following manufacturer instructions.

    Article Title: Salt Stress Effects on Secondary Metabolites of Cotton in Relation to Gene Expression Responsible for Aphid Development
    Article Snippet: The first-strand cDNA was synthesized from 1μg of total RNA with First-Strand Synthesis SuperMix for qRT-PCR kit (Invitrogen). qRT-PCR reactions were performed using the iCycler iQ2 sequence detection system (Bio-Rad, Hercules, CA) with SYBR Green PCR Master Mix (Applied Biosystems). .. Three independent biological replicates of each treatment were performed and normalized to β-actin expression.

    Article Title: Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses
    Article Snippet: After extraction, 1 μg total RNA was used to generate cDNA with the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR kit, which includes both oligo-dT and random primers (Thermo Fisher Scientific, Waltham, MA). .. The relative expression was normalized with the expression of the housekeeping gene 36B4.

    Article Title: Research Resource: The Dexamethasone Transcriptome in Hypothalamic Embryonic Neural Stem Cells
    Article Snippet: The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen) and one universal qPCR reverse primer was used to amplify the poly-dT region of the cDNA T1 along with small RNA-specific forward primers (3′ or 5′). qRT-PCRs were performed on a Bio-Rad CFX qRT-PCR machine using iTaq Universal SYBR Green Supermix (172-5121; Bio-Rad) and primers with efficiencies calculated to be greater than 80% ( ). .. There was high-level expression ( > 330 FPKMs) and no differences observed due to sex or treatment for glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) in our RNA-Seq dataset, and therefore, relative expression was calculated with Gapdh as the reference gene, whereas for microRNAs, U6 served as the reference gene.

    Article Title: miR-10a is aberrantly overexpressed in Nucleophosmin1 mutated acute myeloid leukaemia and its suppression induces cell death
    Article Snippet: .. To confirm miR-10a expression in primary samples and comparison of expression in cell lines, 1 μg of total RNA was used for cDNA synthesis reaction using the NCode microRNA first strand synthesis and qRT-PCR kit (Invitrogen, USA). .. Quantitative real time polymerase chain reaction (qRT-PCR) was performed using the Platinum Sybr Green Taq (Invitrogen, USA) and the Rotorgene RG-3000 thermocycler (Qiagen, Hilden, Germany).

    Derivative Assay:

    Article Title: Highly Sensitive Quantitative Real-Time PCR for the Detection of Plasmodium Liver-Stage Parasite Burden following Low-Dose Sporozoite Challenge
    Article Snippet: Primers and Probes cDNA derived from P. yoelii 18S rRNA (Py18S) was quantified by RT-PCR using a custom dual-labelled probe and custom primers (Geneworks Pty. .. A commercially available qRT-PCR kit (TaqMan® Gene Expression Assays from Applied Biosystems, Life Technologies Australia Pty Ltd., Mulgrave, VIC) was used to amplify mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene for the normalisation of Py18S or PyCytB values.

    Northern Blot:

    Article Title: Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells
    Article Snippet: Paragraph title: Northern blots and qRT-PCR ... The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen).

    Infection:

    Article Title: Non-Coding RNAs are Differentially Expressed by Nocardia brasiliensis in Vitro and in Experimental Actinomycetoma
    Article Snippet: Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) To confirm the presence of the transcripts found by RNA-Seq, qRT-PCR was performed using the NCode™ miRNA First-Strand cDNA Synthesis and qRT-PCR kit (Life Technologies Corporation, cat. no. MIRQ-100) according to the manufacturer´s instructions. .. As starting material we used 1 µg RNA obtained from infection or in vitro growth conditions.

    Generated:

    Article Title: Bacterial Pathogens Activate a Common Inflammatory Pathway through IFN? Regulation of PDCD4
    Article Snippet: .. For miRNA analysis cDNA was generated and qRT-PCR was run using NCode miRNA First-Strand cDNA Synthesis and qRT-PCR Kit (Life Technologies) according to the manufacturers instructions. ..

    Article Title: miR-10a is aberrantly overexpressed in Nucleophosmin1 mutated acute myeloid leukaemia and its suppression induces cell death
    Article Snippet: Hierarchical cluster analysis was then performed using the probe sets identified to be significantly DE and a heat map was generated. .. To confirm miR-10a expression in primary samples and comparison of expression in cell lines, 1 μg of total RNA was used for cDNA synthesis reaction using the NCode microRNA first strand synthesis and qRT-PCR kit (Invitrogen, USA).

    Polymerase Chain Reaction:

    Article Title: Heat Shock Protein 90 (Hsp90) as a Molecular Target for the Development of Novel Drugs Against the Dermatophyte Trichophyton rubrum
    Article Snippet: First-strand cDNA was synthesized using the SuperScriptIII First-Strand Synthesis Super Mix for qRT-PCR kit (Invitrogen). .. Specific primer pairs for each T. rubrum gene were designed using the Primer Express v.3 software (Life Technologies) and are listed in Table . qRT-PCR reactions were carried out in a final volume of 12.5 μL, containing 6.25 μL Power SYBRGreen PCR Master Mix (Life Technologies), 1.0 μL of each primer (hsp20 , 250 nM; hsp60 , 500 nM; hsp70 , 450 nM; hsp70-like , 350 nM; hsp clpa , 300 nM; hsp88-like , 350 nM; hsp90 , 300 nM; cdc37 , 400 nM; hsp ssc1 , 400 nM; hsp78 , 350 nM; hsf1 , 350 nM; pacC , 350 nM), 2.0-μL template cDNA (50 ng), and 3.25-μL ultra-pure water.

    Article Title: Bacterial Pathogens Activate a Common Inflammatory Pathway through IFN? Regulation of PDCD4
    Article Snippet: Quantitative real-time RT-PCR (QRT-PCR) was performed using Power SYBR Green PCR Master Mix in a Step One Plus Thermal Cycler (Applied Biosystems). .. For miRNA analysis cDNA was generated and qRT-PCR was run using NCode miRNA First-Strand cDNA Synthesis and qRT-PCR Kit (Life Technologies) according to the manufacturers instructions.

    Article Title: Astrocytic CCAAT/Enhancer Binding Protein δ Regulates Neuronal Viability and Spatial Learning Ability via miR-135a
    Article Snippet: Specific primers used for the RT-PCR analysis are as follows: for GAPDH (20 cycles and product size: 576 bp), 5′-CCATCACCATCTTCCAGGAG-3′ and 5′-CCTGCTTCACCACCTTCTTG-3′; for THBS1 (28 cycles and product size: 376 bp), 5′-GCTCAGAGTGGATGTTATGG-3′ and 5′-GGGAATACTTCTCTGCAGAG-3′; for Thbs1 (28 cycles and product size: 192 bp), 5′-CCAAAGCCTGCAAGAAAGAC-3′ and 5′-CCTGCTTGTTGCAAACTTGA-3′; for CEBPD (26 cycles and product size: 267 bp), 5′-AGCGCAACAACATCGCCGTG-3′ and 5′-GTCGGGTCTGAGGTATGGGTC-3′; and for Cebpd (26 cycles and product size: 429 bp), 5′-ATCGCTGCAGCTTCCTATGT-3′ and 5′-GGTTAAGCCCGCAAACATTA-3′ The PCR products were separated by electrophoresis in 1 % agarose gels and visualized with ethidium bromide staining. .. Forty cycles were set for qRT-PCR program and the abundances of interested gene expression were calculated following the formula (2 -ΔΔCt ) suggested in the instruction of qRT-PCR kit (Applied Biosystems).

    Article Title: Salt Stress Effects on Secondary Metabolites of Cotton in Relation to Gene Expression Responsible for Aphid Development
    Article Snippet: .. The first-strand cDNA was synthesized from 1μg of total RNA with First-Strand Synthesis SuperMix for qRT-PCR kit (Invitrogen). qRT-PCR reactions were performed using the iCycler iQ2 sequence detection system (Bio-Rad, Hercules, CA) with SYBR Green PCR Master Mix (Applied Biosystems). .. The PCR parameters were 95°C for 3min; 40 cycles at 95°C for 10s, at annealing temperatures of 60°C for 10s, and 72°C for 10s.

    Article Title: Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses
    Article Snippet: After extraction, 1 μg total RNA was used to generate cDNA with the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR kit, which includes both oligo-dT and random primers (Thermo Fisher Scientific, Waltham, MA). .. Quantitative reverse transcription PCR (qRT-PCR) of the indicated genes ( ) was performed using SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA) and the Applied Biosystems 7300 Fast real-time PCR system and software.

    Article Title: Novel Tissue-Specific Mechanism of Regulation of Angiogenesis and Cancer Growth in Response to Hyperglycemia
    Article Snippet: To measure miRNA levels, 1 μg of total RNA was first polyadenylated followed by first‐strand cDNA synthesis using the protocol for NCode miRNA First‐Strand cDNA Synthesis and a qRT‐PCR kit (Invitrogen). .. The miRNA sequence‐specific primers used for PCR were purchased from Invitrogen, and the amplification cycles were set according to the instructions outlined in the kit.

    Article Title: Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells
    Article Snippet: The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen). .. PCR was performed with input, IgG-associated, and YB-1–associated RNAs.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Highly Sensitive Quantitative Real-Time PCR for the Detection of Plasmodium Liver-Stage Parasite Burden following Low-Dose Sporozoite Challenge
    Article Snippet: Primers and Probes cDNA derived from P. yoelii 18S rRNA (Py18S) was quantified by RT-PCR using a custom dual-labelled probe and custom primers (Geneworks Pty. .. A commercially available qRT-PCR kit (TaqMan® Gene Expression Assays from Applied Biosystems, Life Technologies Australia Pty Ltd., Mulgrave, VIC) was used to amplify mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene for the normalisation of Py18S or PyCytB values.

    Article Title: Astrocytic CCAAT/Enhancer Binding Protein δ Regulates Neuronal Viability and Spatial Learning Ability via miR-135a
    Article Snippet: Specific primers used for the RT-PCR analysis are as follows: for GAPDH (20 cycles and product size: 576 bp), 5′-CCATCACCATCTTCCAGGAG-3′ and 5′-CCTGCTTCACCACCTTCTTG-3′; for THBS1 (28 cycles and product size: 376 bp), 5′-GCTCAGAGTGGATGTTATGG-3′ and 5′-GGGAATACTTCTCTGCAGAG-3′; for Thbs1 (28 cycles and product size: 192 bp), 5′-CCAAAGCCTGCAAGAAAGAC-3′ and 5′-CCTGCTTGTTGCAAACTTGA-3′; for CEBPD (26 cycles and product size: 267 bp), 5′-AGCGCAACAACATCGCCGTG-3′ and 5′-GTCGGGTCTGAGGTATGGGTC-3′; and for Cebpd (26 cycles and product size: 429 bp), 5′-ATCGCTGCAGCTTCCTATGT-3′ and 5′-GGTTAAGCCCGCAAACATTA-3′ The PCR products were separated by electrophoresis in 1 % agarose gels and visualized with ethidium bromide staining. .. Forty cycles were set for qRT-PCR program and the abundances of interested gene expression were calculated following the formula (2 -ΔΔCt ) suggested in the instruction of qRT-PCR kit (Applied Biosystems).

    Article Title: Aedes aegypti microRNA miR-2b regulates ubiquitin-related modifier to control chikungunya virus replication
    Article Snippet: For miRNAs, 1 µg of RNA was polyadenylated, reverse transcribed, and quantified by qRT-PCR using NCode miRNA First Strand cDNA Synthesis and qRT-PCR kit (Thermo Fisher Scientific, MA). qRT-PCR reactions were set up using 1:10 diluted cDNA as template following manufacturer instructions. .. For other transcripts, cDNA was used directly for quantitative RT-PCR using QuantiTect SYBR Green RT-PCR Kit (Qiagen, Germany).

    Article Title: Non-Coding RNAs are Differentially Expressed by Nocardia brasiliensis in Vitro and in Experimental Actinomycetoma
    Article Snippet: .. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) To confirm the presence of the transcripts found by RNA-Seq, qRT-PCR was performed using the NCode™ miRNA First-Strand cDNA Synthesis and qRT-PCR kit (Life Technologies Corporation, cat. no. MIRQ-100) according to the manufacturer´s instructions. .. As starting material we used 1 µg RNA obtained from infection or in vitro growth conditions.

    Article Title: CCR7 promote lymph node metastasis via regulating VEGF-C/D-R3 pathway in lung adenocarcinoma
    Article Snippet: Quantitative reverse transcriptase-polymerase chain reaction Total RNA was extracted by the RNAiso plus (Invitrogen Co), cDNA was transcribed from mRNA with Prime Script RT-PCR Kit and Oligo dT Primer (Invitrogen Co). .. The qRT-PCR kit was provided by Invitrogen.

    Article Title: Novel Tissue-Specific Mechanism of Regulation of Angiogenesis and Cancer Growth in Response to Hyperglycemia
    Article Snippet: Two micrograms of the total RNA was used to synthesize first‐strand cDNA using reagents and the protocol from the Superscript First Strand Synthesis System for RT‐PCR (Invitrogen). .. To measure miRNA levels, 1 μg of total RNA was first polyadenylated followed by first‐strand cDNA synthesis using the protocol for NCode miRNA First‐Strand cDNA Synthesis and a qRT‐PCR kit (Invitrogen).

    Binding Assay:

    Article Title: Salt Stress Effects on Secondary Metabolites of Cotton in Relation to Gene Expression Responsible for Aphid Development
    Article Snippet: Quantitative Real-Time PCR qRT-PCR was used to determine the transcript levels of some important genes including P450, NADH, lipid biosynthesis, juvenile hormone binding protein (Jhbp) and ecdyson. .. The first-strand cDNA was synthesized from 1μg of total RNA with First-Strand Synthesis SuperMix for qRT-PCR kit (Invitrogen). qRT-PCR reactions were performed using the iCycler iQ2 sequence detection system (Bio-Rad, Hercules, CA) with SYBR Green PCR Master Mix (Applied Biosystems).

    RNA Sequencing Assay:

    Article Title: Non-Coding RNAs are Differentially Expressed by Nocardia brasiliensis in Vitro and in Experimental Actinomycetoma
    Article Snippet: .. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) To confirm the presence of the transcripts found by RNA-Seq, qRT-PCR was performed using the NCode™ miRNA First-Strand cDNA Synthesis and qRT-PCR kit (Life Technologies Corporation, cat. no. MIRQ-100) according to the manufacturer´s instructions. .. As starting material we used 1 µg RNA obtained from infection or in vitro growth conditions.

    Article Title: Research Resource: The Dexamethasone Transcriptome in Hypothalamic Embryonic Neural Stem Cells
    Article Snippet: The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen) and one universal qPCR reverse primer was used to amplify the poly-dT region of the cDNA T1 along with small RNA-specific forward primers (3′ or 5′). qRT-PCRs were performed on a Bio-Rad CFX qRT-PCR machine using iTaq Universal SYBR Green Supermix (172-5121; Bio-Rad) and primers with efficiencies calculated to be greater than 80% ( ). .. There was high-level expression ( > 330 FPKMs) and no differences observed due to sex or treatment for glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) in our RNA-Seq dataset, and therefore, relative expression was calculated with Gapdh as the reference gene, whereas for microRNAs, U6 served as the reference gene.

    Isolation:

    Article Title: Heat Shock Protein 90 (Hsp90) as a Molecular Target for the Development of Novel Drugs Against the Dermatophyte Trichophyton rubrum
    Article Snippet: Gene Expression Analysis Total RNA was isolated from frozen mycelia using Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare). .. First-strand cDNA was synthesized using the SuperScriptIII First-Strand Synthesis Super Mix for qRT-PCR kit (Invitrogen).

    Article Title: Astrocytic CCAAT/Enhancer Binding Protein δ Regulates Neuronal Viability and Spatial Learning Ability via miR-135a
    Article Snippet: Paragraph title: RNA Isolation, Reverse Transcription, and Quantitative Real-Time PCR ... Forty cycles were set for qRT-PCR program and the abundances of interested gene expression were calculated following the formula (2 -ΔΔCt ) suggested in the instruction of qRT-PCR kit (Applied Biosystems).

    Article Title: Research Resource: The Dexamethasone Transcriptome in Hypothalamic Embryonic Neural Stem Cells
    Article Snippet: Cells were harvested in TRIzol and processed for RNA isolation using chloroform extraction. cDNA synthesis was performed using iScript Select cDNA Synthesis kit (170-8897; Bio-Rad). .. The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen) and one universal qPCR reverse primer was used to amplify the poly-dT region of the cDNA T1 along with small RNA-specific forward primers (3′ or 5′). qRT-PCRs were performed on a Bio-Rad CFX qRT-PCR machine using iTaq Universal SYBR Green Supermix (172-5121; Bio-Rad) and primers with efficiencies calculated to be greater than 80% ( ).

    Labeling:

    Article Title: Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells
    Article Snippet: LNA or DNA oligonucleotide probes were 3′-end labeled with digoxigenin (DIG) using an End Tailing Kit (Roche). .. The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen).

    Purification:

    Article Title: Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells
    Article Snippet: RNA obtained from the immunoprecipitation experiments were size fractionated and gel purified. .. The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen).

    Article Title: Research Resource: The Dexamethasone Transcriptome in Hypothalamic Embryonic Neural Stem Cells
    Article Snippet: For microRNAs, total RNAs were size fractionated and gel purified ( ). .. The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen) and one universal qPCR reverse primer was used to amplify the poly-dT region of the cDNA T1 along with small RNA-specific forward primers (3′ or 5′). qRT-PCRs were performed on a Bio-Rad CFX qRT-PCR machine using iTaq Universal SYBR Green Supermix (172-5121; Bio-Rad) and primers with efficiencies calculated to be greater than 80% ( ).

    Sequencing:

    Article Title: Highly Sensitive Quantitative Real-Time PCR for the Detection of Plasmodium Liver-Stage Parasite Burden following Low-Dose Sporozoite Challenge
    Article Snippet: Amplification with these primers generates a 207bp fragment ( ) that contains 143 mismatches (41.6% homology) with the homologous mouse cytochrome B mtDNA sequence (Cyb561; ). .. A commercially available qRT-PCR kit (TaqMan® Gene Expression Assays from Applied Biosystems, Life Technologies Australia Pty Ltd., Mulgrave, VIC) was used to amplify mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene for the normalisation of Py18S or PyCytB values.

    Article Title: Salt Stress Effects on Secondary Metabolites of Cotton in Relation to Gene Expression Responsible for Aphid Development
    Article Snippet: .. The first-strand cDNA was synthesized from 1μg of total RNA with First-Strand Synthesis SuperMix for qRT-PCR kit (Invitrogen). qRT-PCR reactions were performed using the iCycler iQ2 sequence detection system (Bio-Rad, Hercules, CA) with SYBR Green PCR Master Mix (Applied Biosystems). .. The PCR parameters were 95°C for 3min; 40 cycles at 95°C for 10s, at annealing temperatures of 60°C for 10s, and 72°C for 10s.

    Article Title: Novel Tissue-Specific Mechanism of Regulation of Angiogenesis and Cancer Growth in Response to Hyperglycemia
    Article Snippet: To measure miRNA levels, 1 μg of total RNA was first polyadenylated followed by first‐strand cDNA synthesis using the protocol for NCode miRNA First‐Strand cDNA Synthesis and a qRT‐PCR kit (Invitrogen). .. The miRNA sequence‐specific primers used for PCR were purchased from Invitrogen, and the amplification cycles were set according to the instructions outlined in the kit.

    Software:

    Article Title: Highly Sensitive Quantitative Real-Time PCR for the Detection of Plasmodium Liver-Stage Parasite Burden following Low-Dose Sporozoite Challenge
    Article Snippet: P. yoelii cytochrome B mtDNA (PyCytB) specific primers and probe were based on the previously published P. yoelii (17XNL) CytB sequence (PY00774; NCBI Gene ID: 3792183) and designed using the Primer3 software (http://simgene.com/Primer3). .. A commercially available qRT-PCR kit (TaqMan® Gene Expression Assays from Applied Biosystems, Life Technologies Australia Pty Ltd., Mulgrave, VIC) was used to amplify mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene for the normalisation of Py18S or PyCytB values.

    Article Title: Heat Shock Protein 90 (Hsp90) as a Molecular Target for the Development of Novel Drugs Against the Dermatophyte Trichophyton rubrum
    Article Snippet: First-strand cDNA was synthesized using the SuperScriptIII First-Strand Synthesis Super Mix for qRT-PCR kit (Invitrogen). .. Specific primer pairs for each T. rubrum gene were designed using the Primer Express v.3 software (Life Technologies) and are listed in Table . qRT-PCR reactions were carried out in a final volume of 12.5 μL, containing 6.25 μL Power SYBRGreen PCR Master Mix (Life Technologies), 1.0 μL of each primer (hsp20 , 250 nM; hsp60 , 500 nM; hsp70 , 450 nM; hsp70-like , 350 nM; hsp clpa , 300 nM; hsp88-like , 350 nM; hsp90 , 300 nM; cdc37 , 400 nM; hsp ssc1 , 400 nM; hsp78 , 350 nM; hsf1 , 350 nM; pacC , 350 nM), 2.0-μL template cDNA (50 ng), and 3.25-μL ultra-pure water.

    Article Title: Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses
    Article Snippet: After extraction, 1 μg total RNA was used to generate cDNA with the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR kit, which includes both oligo-dT and random primers (Thermo Fisher Scientific, Waltham, MA). .. Quantitative reverse transcription PCR (qRT-PCR) of the indicated genes ( ) was performed using SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA) and the Applied Biosystems 7300 Fast real-time PCR system and software.

    Real-time Polymerase Chain Reaction:

    Article Title: Astrocytic CCAAT/Enhancer Binding Protein δ Regulates Neuronal Viability and Spatial Learning Ability via miR-135a
    Article Snippet: Paragraph title: RNA Isolation, Reverse Transcription, and Quantitative Real-Time PCR ... Forty cycles were set for qRT-PCR program and the abundances of interested gene expression were calculated following the formula (2 -ΔΔCt ) suggested in the instruction of qRT-PCR kit (Applied Biosystems).

    Article Title: Salt Stress Effects on Secondary Metabolites of Cotton in Relation to Gene Expression Responsible for Aphid Development
    Article Snippet: Paragraph title: Quantitative Real-Time PCR ... The first-strand cDNA was synthesized from 1μg of total RNA with First-Strand Synthesis SuperMix for qRT-PCR kit (Invitrogen). qRT-PCR reactions were performed using the iCycler iQ2 sequence detection system (Bio-Rad, Hercules, CA) with SYBR Green PCR Master Mix (Applied Biosystems).

    Article Title: CCR7 promote lymph node metastasis via regulating VEGF-C/D-R3 pathway in lung adenocarcinoma
    Article Snippet: The mRNA level of CCR7, VEGF-R3, VEGF-C, VEGF-D were measured by Real-time quantitative PCR Thermal Cycler Dice Real Time System was used to amplification react the cDNA. .. The qRT-PCR kit was provided by Invitrogen.

    Article Title: Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses
    Article Snippet: After extraction, 1 μg total RNA was used to generate cDNA with the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR kit, which includes both oligo-dT and random primers (Thermo Fisher Scientific, Waltham, MA). .. Quantitative reverse transcription PCR (qRT-PCR) of the indicated genes ( ) was performed using SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA) and the Applied Biosystems 7300 Fast real-time PCR system and software.

    Article Title: Novel Tissue-Specific Mechanism of Regulation of Angiogenesis and Cancer Growth in Response to Hyperglycemia
    Article Snippet: To measure miRNA levels, 1 μg of total RNA was first polyadenylated followed by first‐strand cDNA synthesis using the protocol for NCode miRNA First‐Strand cDNA Synthesis and a qRT‐PCR kit (Invitrogen). .. Real‐time PCR amplification was performed with reagents from the same kit.

    Article Title: Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells
    Article Snippet: The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen). .. For validation of small RNAs, one universal qPCR reverse primer was used to amplify the poly-dT region of the cDNA along with small RNA specific forward primers ( ).

    Article Title: Research Resource: The Dexamethasone Transcriptome in Hypothalamic Embryonic Neural Stem Cells
    Article Snippet: .. The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen) and one universal qPCR reverse primer was used to amplify the poly-dT region of the cDNA T1 along with small RNA-specific forward primers (3′ or 5′). qRT-PCRs were performed on a Bio-Rad CFX qRT-PCR machine using iTaq Universal SYBR Green Supermix (172-5121; Bio-Rad) and primers with efficiencies calculated to be greater than 80% ( ). .. There was high-level expression ( > 330 FPKMs) and no differences observed due to sex or treatment for glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) in our RNA-Seq dataset, and therefore, relative expression was calculated with Gapdh as the reference gene, whereas for microRNAs, U6 served as the reference gene.

    Article Title: miR-10a is aberrantly overexpressed in Nucleophosmin1 mutated acute myeloid leukaemia and its suppression induces cell death
    Article Snippet: To confirm miR-10a expression in primary samples and comparison of expression in cell lines, 1 μg of total RNA was used for cDNA synthesis reaction using the NCode microRNA first strand synthesis and qRT-PCR kit (Invitrogen, USA). .. Quantitative real time polymerase chain reaction (qRT-PCR) was performed using the Platinum Sybr Green Taq (Invitrogen, USA) and the Rotorgene RG-3000 thermocycler (Qiagen, Hilden, Germany).

    RNA Extraction:

    Article Title: Heat Shock Protein 90 (Hsp90) as a Molecular Target for the Development of Novel Drugs Against the Dermatophyte Trichophyton rubrum
    Article Snippet: First-strand cDNA was synthesized using the SuperScriptIII First-Strand Synthesis Super Mix for qRT-PCR kit (Invitrogen). .. Both RNA extraction and cDNA synthesis were performed according to the manufacturer’s recommendations.

    Article Title: MicroRNAs in metamorphic and non-metamorphic transitions in hemimetabolan insect metamorphosis
    Article Snippet: Whole body sampling and total RNA extraction was carried out as in the library construction. .. An amount of 400 ng of total RNA was reverse transcribed with the NCode miRNA first-strand synthesis and qRT-PCR kit (Invitrogen), following the manufacturer protocol.

    Article Title: Astrocytic CCAAT/Enhancer Binding Protein δ Regulates Neuronal Viability and Spatial Learning Ability via miR-135a
    Article Snippet: RNA Isolation, Reverse Transcription, and Quantitative Real-Time PCR Total RNAs were isolated using the TRIzol RNA extraction reagent and subjected to reverse transcription with SuperScriptTM III. .. Forty cycles were set for qRT-PCR program and the abundances of interested gene expression were calculated following the formula (2 -ΔΔCt ) suggested in the instruction of qRT-PCR kit (Applied Biosystems).

    In Vitro:

    Article Title: Non-Coding RNAs are Differentially Expressed by Nocardia brasiliensis in Vitro and in Experimental Actinomycetoma
    Article Snippet: Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) To confirm the presence of the transcripts found by RNA-Seq, qRT-PCR was performed using the NCode™ miRNA First-Strand cDNA Synthesis and qRT-PCR kit (Life Technologies Corporation, cat. no. MIRQ-100) according to the manufacturer´s instructions. .. As starting material we used 1 µg RNA obtained from infection or in vitro growth conditions.

    Electrophoresis:

    Article Title: Astrocytic CCAAT/Enhancer Binding Protein δ Regulates Neuronal Viability and Spatial Learning Ability via miR-135a
    Article Snippet: Specific primers used for the RT-PCR analysis are as follows: for GAPDH (20 cycles and product size: 576 bp), 5′-CCATCACCATCTTCCAGGAG-3′ and 5′-CCTGCTTCACCACCTTCTTG-3′; for THBS1 (28 cycles and product size: 376 bp), 5′-GCTCAGAGTGGATGTTATGG-3′ and 5′-GGGAATACTTCTCTGCAGAG-3′; for Thbs1 (28 cycles and product size: 192 bp), 5′-CCAAAGCCTGCAAGAAAGAC-3′ and 5′-CCTGCTTGTTGCAAACTTGA-3′; for CEBPD (26 cycles and product size: 267 bp), 5′-AGCGCAACAACATCGCCGTG-3′ and 5′-GTCGGGTCTGAGGTATGGGTC-3′; and for Cebpd (26 cycles and product size: 429 bp), 5′-ATCGCTGCAGCTTCCTATGT-3′ and 5′-GGTTAAGCCCGCAAACATTA-3′ The PCR products were separated by electrophoresis in 1 % agarose gels and visualized with ethidium bromide staining. .. Forty cycles were set for qRT-PCR program and the abundances of interested gene expression were calculated following the formula (2 -ΔΔCt ) suggested in the instruction of qRT-PCR kit (Applied Biosystems).

    Sampling:

    Article Title: MicroRNAs in metamorphic and non-metamorphic transitions in hemimetabolan insect metamorphosis
    Article Snippet: Whole body sampling and total RNA extraction was carried out as in the library construction. .. An amount of 400 ng of total RNA was reverse transcribed with the NCode miRNA first-strand synthesis and qRT-PCR kit (Invitrogen), following the manufacturer protocol.

    Immunoprecipitation:

    Article Title: Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells
    Article Snippet: RNA obtained from the immunoprecipitation experiments were size fractionated and gel purified. .. The NCode VILO cDNA synthesis and qRT-PCR kit was used to polyadenylate the resulting RNA and oligo(DT) primers were used to synthesize the first strand cDNA (Invitrogen).

    Staining:

    Article Title: Astrocytic CCAAT/Enhancer Binding Protein δ Regulates Neuronal Viability and Spatial Learning Ability via miR-135a
    Article Snippet: Specific primers used for the RT-PCR analysis are as follows: for GAPDH (20 cycles and product size: 576 bp), 5′-CCATCACCATCTTCCAGGAG-3′ and 5′-CCTGCTTCACCACCTTCTTG-3′; for THBS1 (28 cycles and product size: 376 bp), 5′-GCTCAGAGTGGATGTTATGG-3′ and 5′-GGGAATACTTCTCTGCAGAG-3′; for Thbs1 (28 cycles and product size: 192 bp), 5′-CCAAAGCCTGCAAGAAAGAC-3′ and 5′-CCTGCTTGTTGCAAACTTGA-3′; for CEBPD (26 cycles and product size: 267 bp), 5′-AGCGCAACAACATCGCCGTG-3′ and 5′-GTCGGGTCTGAGGTATGGGTC-3′; and for Cebpd (26 cycles and product size: 429 bp), 5′-ATCGCTGCAGCTTCCTATGT-3′ and 5′-GGTTAAGCCCGCAAACATTA-3′ The PCR products were separated by electrophoresis in 1 % agarose gels and visualized with ethidium bromide staining. .. Forty cycles were set for qRT-PCR program and the abundances of interested gene expression were calculated following the formula (2 -ΔΔCt ) suggested in the instruction of qRT-PCR kit (Applied Biosystems).

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  • 90
    Thermo Fisher express one step superscript qrt pcr kit
    Down-regulation of c-MYC pathway by ibrutinib±GSI in primary B-CLL cell cultures Patients’ derived B-CLL cells co-cultured with stromal cells were exposed to Ibrutinib±GSI for 24 hours or were grown untreated in suspension as control. In (A) , Western blotting analyses of c-MYC protein levels are shown for representative primary B-CLL patients. For clarity, tubulin is shown as loading control for one patient. In (B) , levels of c-MYC mRNA were analyzed by <t>qRT-PCR</t> and are expressed as fold of modulation with respect to the untreated B-CLL cultures grown in suspension set at 1. Results are reported as mean±SD of four independent experiments, performed in duplicate. The asterisk indicates p
    Express One Step Superscript Qrt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Discovery of an IFN-inducible subclass of ERVs. ( a ) Immunoblot of pTBK1, TBK1, IKKε, pIRF3, pERK, ERK, pAKT, AKT and tubulin levels in H69 and H69M cells after 24 or 72 h culture. ( b ) Log-2 fold change cytokine/chemokine differences between H69M/H69 CM. ( c ) H E and pTBK1 IHC of a patient-derived SCLC brain metastasis. Scale bar indicates 20 μm. ( d ) Isotype control versus PD-L1 or CD44 surface expression on H69 and H69M cells ± 200 ng/mL 24 h IFNγ stimulation (representative of n=3 biological replicates). ( e ) Immunoblot of pTBK1, TBK1, pERK, ERK, pAKT, AKT and β-actin levels in H69, H69M, H69M-PD-L1 low , and H69M-PD-L1 high cells. ( f ) Log-2 fold change cytokine/chemokines differences between H69M-PD-L1 high or H69M-PD-L1 low /H69 CM. ( g ) <t>qRT-PCR</t> of ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low of previously published ERVs. Error bars are mean ± s.e.m of n=3 biological replicates. TRIM22 promoter and antisense orientation of MLT1C49 in the 3′UTR are represented below the qRT-PCR graph. ( h ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 low cells transfected with pLX-307-GFP control or pLX_307-MLT1C49 construct for 72h. Mean ± s.e.m of n=3 biological replicates shown. ( i ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 high cells transfected with scrambled negative control siRNA or siRNAs specific for MLT1C49. Mean ± s.e.m of n=3 biological replicates shown. ( j ) Overlap of 3′UTR antisense ERVs with H69M upregulated genes (log-2 fold change relative to H69 > 2) and table showing the fold change in expression of these genes/ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low cells. ( k ) Immunoblot of STING, MAVS, pTBK1, TBK1, pIRF3, IRF3, E-Cadherin, Vimentin and β-actin levels in H69M cells after CRISPR mediated deletion of MAVS and/or STING. ( l ) Log-2 fold change cytokine/chemokine differences in CM from H69M cells after CRISPR mediated deletion of MAVS and/or STING compared to sgCTRL (Scramble). ( m ) CXCL10 Luminex absolute levels (pg/mL) in Scramble, STING KO, MAVS KO and double KO (dKO) H69M cells. Mean ± s.e.m of n=2 biological replicates shown. *p
    Qrt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Weight change, survival, and viral RNA levels in RVFV-inoculated humanized mice. (A) Weight change (mean ± SD) and (B) survival in unengrafted NSG-SGM3 (NSGS) mice and Hu-NSG-SGM3 mice inoculated intramuscularly with 10 4 TCID 50 (Hi) or 10 1 TCID 50 (Lo) of RVFV. Humanized mice were inoculated either 13 or 19 weeks (wk) post engraftment, as indicated. (C) Viral RNA genome copy number (per μL of RNA) normalized to 18S in blood, liver, spleen, and brain collected at time of terminal euthanasia (NSGS mice: 2–3 DPI for Hi or 3–4 DPI for Lo; Hu-NSG-SGM3 mice: 2 DPI for Hi or 3 DPI for Lo) determined by <t>qRT-PCR</t> (mean ± SD). Blood was obtained from all but 3 mice (Lo-13-wk-1 [ID 5082–15], and one mouse each in the Hi- and Lo-NSGS groups). Significant at confidence of *p ≦ 0.05 (NSGS vs. Hu-NSG-SGM3 at same dose); **p
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    Image Search Results


    Down-regulation of c-MYC pathway by ibrutinib±GSI in primary B-CLL cell cultures Patients’ derived B-CLL cells co-cultured with stromal cells were exposed to Ibrutinib±GSI for 24 hours or were grown untreated in suspension as control. In (A) , Western blotting analyses of c-MYC protein levels are shown for representative primary B-CLL patients. For clarity, tubulin is shown as loading control for one patient. In (B) , levels of c-MYC mRNA were analyzed by qRT-PCR and are expressed as fold of modulation with respect to the untreated B-CLL cultures grown in suspension set at 1. Results are reported as mean±SD of four independent experiments, performed in duplicate. The asterisk indicates p

    Journal: Oncotarget

    Article Title: The γ-secretase inhibitors enhance the anti-leukemic activity of ibrutinib in B-CLL cells

    doi: 10.18632/oncotarget.19494

    Figure Lengend Snippet: Down-regulation of c-MYC pathway by ibrutinib±GSI in primary B-CLL cell cultures Patients’ derived B-CLL cells co-cultured with stromal cells were exposed to Ibrutinib±GSI for 24 hours or were grown untreated in suspension as control. In (A) , Western blotting analyses of c-MYC protein levels are shown for representative primary B-CLL patients. For clarity, tubulin is shown as loading control for one patient. In (B) , levels of c-MYC mRNA were analyzed by qRT-PCR and are expressed as fold of modulation with respect to the untreated B-CLL cultures grown in suspension set at 1. Results are reported as mean±SD of four independent experiments, performed in duplicate. The asterisk indicates p

    Article Snippet: For each sample, total RNA (300 ng) was transcribed into cDNA and amplified using the Express One-Step Superscript qRT-PCR Kit, universal (Thermo Fisher Scientific, Rockford, IL).

    Techniques: Derivative Assay, Cell Culture, Western Blot, Quantitative RT-PCR

    Discovery of an IFN-inducible subclass of ERVs. ( a ) Immunoblot of pTBK1, TBK1, IKKε, pIRF3, pERK, ERK, pAKT, AKT and tubulin levels in H69 and H69M cells after 24 or 72 h culture. ( b ) Log-2 fold change cytokine/chemokine differences between H69M/H69 CM. ( c ) H E and pTBK1 IHC of a patient-derived SCLC brain metastasis. Scale bar indicates 20 μm. ( d ) Isotype control versus PD-L1 or CD44 surface expression on H69 and H69M cells ± 200 ng/mL 24 h IFNγ stimulation (representative of n=3 biological replicates). ( e ) Immunoblot of pTBK1, TBK1, pERK, ERK, pAKT, AKT and β-actin levels in H69, H69M, H69M-PD-L1 low , and H69M-PD-L1 high cells. ( f ) Log-2 fold change cytokine/chemokines differences between H69M-PD-L1 high or H69M-PD-L1 low /H69 CM. ( g ) qRT-PCR of ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low of previously published ERVs. Error bars are mean ± s.e.m of n=3 biological replicates. TRIM22 promoter and antisense orientation of MLT1C49 in the 3′UTR are represented below the qRT-PCR graph. ( h ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 low cells transfected with pLX-307-GFP control or pLX_307-MLT1C49 construct for 72h. Mean ± s.e.m of n=3 biological replicates shown. ( i ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 high cells transfected with scrambled negative control siRNA or siRNAs specific for MLT1C49. Mean ± s.e.m of n=3 biological replicates shown. ( j ) Overlap of 3′UTR antisense ERVs with H69M upregulated genes (log-2 fold change relative to H69 > 2) and table showing the fold change in expression of these genes/ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low cells. ( k ) Immunoblot of STING, MAVS, pTBK1, TBK1, pIRF3, IRF3, E-Cadherin, Vimentin and β-actin levels in H69M cells after CRISPR mediated deletion of MAVS and/or STING. ( l ) Log-2 fold change cytokine/chemokine differences in CM from H69M cells after CRISPR mediated deletion of MAVS and/or STING compared to sgCTRL (Scramble). ( m ) CXCL10 Luminex absolute levels (pg/mL) in Scramble, STING KO, MAVS KO and double KO (dKO) H69M cells. Mean ± s.e.m of n=2 biological replicates shown. *p

    Journal: Nature medicine

    Article Title: Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses

    doi: 10.1038/s41591-018-0116-5

    Figure Lengend Snippet: Discovery of an IFN-inducible subclass of ERVs. ( a ) Immunoblot of pTBK1, TBK1, IKKε, pIRF3, pERK, ERK, pAKT, AKT and tubulin levels in H69 and H69M cells after 24 or 72 h culture. ( b ) Log-2 fold change cytokine/chemokine differences between H69M/H69 CM. ( c ) H E and pTBK1 IHC of a patient-derived SCLC brain metastasis. Scale bar indicates 20 μm. ( d ) Isotype control versus PD-L1 or CD44 surface expression on H69 and H69M cells ± 200 ng/mL 24 h IFNγ stimulation (representative of n=3 biological replicates). ( e ) Immunoblot of pTBK1, TBK1, pERK, ERK, pAKT, AKT and β-actin levels in H69, H69M, H69M-PD-L1 low , and H69M-PD-L1 high cells. ( f ) Log-2 fold change cytokine/chemokines differences between H69M-PD-L1 high or H69M-PD-L1 low /H69 CM. ( g ) qRT-PCR of ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low of previously published ERVs. Error bars are mean ± s.e.m of n=3 biological replicates. TRIM22 promoter and antisense orientation of MLT1C49 in the 3′UTR are represented below the qRT-PCR graph. ( h ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 low cells transfected with pLX-307-GFP control or pLX_307-MLT1C49 construct for 72h. Mean ± s.e.m of n=3 biological replicates shown. ( i ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 high cells transfected with scrambled negative control siRNA or siRNAs specific for MLT1C49. Mean ± s.e.m of n=3 biological replicates shown. ( j ) Overlap of 3′UTR antisense ERVs with H69M upregulated genes (log-2 fold change relative to H69 > 2) and table showing the fold change in expression of these genes/ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low cells. ( k ) Immunoblot of STING, MAVS, pTBK1, TBK1, pIRF3, IRF3, E-Cadherin, Vimentin and β-actin levels in H69M cells after CRISPR mediated deletion of MAVS and/or STING. ( l ) Log-2 fold change cytokine/chemokine differences in CM from H69M cells after CRISPR mediated deletion of MAVS and/or STING compared to sgCTRL (Scramble). ( m ) CXCL10 Luminex absolute levels (pg/mL) in Scramble, STING KO, MAVS KO and double KO (dKO) H69M cells. Mean ± s.e.m of n=2 biological replicates shown. *p

    Article Snippet: After extraction, 1 μg total RNA was used to generate cDNA with the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR kit, which includes both oligo-dT and random primers (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Immunohistochemistry, Derivative Assay, Expressing, Quantitative RT-PCR, Transfection, Construct, Negative Control, CRISPR, Luminex

    Expression of SPARCS-containing genes across cancers. ( a ) ssGSEA of SPARCS-gene containing signature across TCGA (n=3602 tumors) and significantly associated gene sets grouped based on biological annotations. IC = information coefficient. FDR = false discovery rate. ( b ) Intersection of top 1000 genes co-regulated with SPARCS-containing gene signature in TCGA and CCLE datasets. MHC class I pathway genes in top 40 highlighted in red, EMT related genes in blue and immune evasion markers in green. ( c ) Distribution of high versus low SPARCS-containing gene expression by TCGA cancer histology. ( d ) Immunoblot of AXL, MET, Vimentin, STING, MAVS and β-actin levels in cell lines with high, intermediate, or low SPARCS gene signature expression after 72 h culture. ( e ) qRT-PCR of MLT1C49 , CXCL10, PD-L1 and CD44 in SPARCS high and SPARCS low cell lines ± IFNγ 10 min pulse - 24 h chase. p values indicated for comparison of SPARCS high versus SPARCS low groups. Mean ± s.e.m of n=2 biological replicates shown (Two-tailed Mann-Whitney U test). *p

    Journal: Nature medicine

    Article Title: Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses

    doi: 10.1038/s41591-018-0116-5

    Figure Lengend Snippet: Expression of SPARCS-containing genes across cancers. ( a ) ssGSEA of SPARCS-gene containing signature across TCGA (n=3602 tumors) and significantly associated gene sets grouped based on biological annotations. IC = information coefficient. FDR = false discovery rate. ( b ) Intersection of top 1000 genes co-regulated with SPARCS-containing gene signature in TCGA and CCLE datasets. MHC class I pathway genes in top 40 highlighted in red, EMT related genes in blue and immune evasion markers in green. ( c ) Distribution of high versus low SPARCS-containing gene expression by TCGA cancer histology. ( d ) Immunoblot of AXL, MET, Vimentin, STING, MAVS and β-actin levels in cell lines with high, intermediate, or low SPARCS gene signature expression after 72 h culture. ( e ) qRT-PCR of MLT1C49 , CXCL10, PD-L1 and CD44 in SPARCS high and SPARCS low cell lines ± IFNγ 10 min pulse - 24 h chase. p values indicated for comparison of SPARCS high versus SPARCS low groups. Mean ± s.e.m of n=2 biological replicates shown (Two-tailed Mann-Whitney U test). *p

    Article Snippet: After extraction, 1 μg total RNA was used to generate cDNA with the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR kit, which includes both oligo-dT and random primers (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

    Weight change, survival, and viral RNA levels in RVFV-inoculated humanized mice. (A) Weight change (mean ± SD) and (B) survival in unengrafted NSG-SGM3 (NSGS) mice and Hu-NSG-SGM3 mice inoculated intramuscularly with 10 4 TCID 50 (Hi) or 10 1 TCID 50 (Lo) of RVFV. Humanized mice were inoculated either 13 or 19 weeks (wk) post engraftment, as indicated. (C) Viral RNA genome copy number (per μL of RNA) normalized to 18S in blood, liver, spleen, and brain collected at time of terminal euthanasia (NSGS mice: 2–3 DPI for Hi or 3–4 DPI for Lo; Hu-NSG-SGM3 mice: 2 DPI for Hi or 3 DPI for Lo) determined by qRT-PCR (mean ± SD). Blood was obtained from all but 3 mice (Lo-13-wk-1 [ID 5082–15], and one mouse each in the Hi- and Lo-NSGS groups). Significant at confidence of *p ≦ 0.05 (NSGS vs. Hu-NSG-SGM3 at same dose); **p

    Journal: PLoS ONE

    Article Title: Human immune cell engraftment does not alter development of severe acute Rift Valley fever in mice

    doi: 10.1371/journal.pone.0201104

    Figure Lengend Snippet: Weight change, survival, and viral RNA levels in RVFV-inoculated humanized mice. (A) Weight change (mean ± SD) and (B) survival in unengrafted NSG-SGM3 (NSGS) mice and Hu-NSG-SGM3 mice inoculated intramuscularly with 10 4 TCID 50 (Hi) or 10 1 TCID 50 (Lo) of RVFV. Humanized mice were inoculated either 13 or 19 weeks (wk) post engraftment, as indicated. (C) Viral RNA genome copy number (per μL of RNA) normalized to 18S in blood, liver, spleen, and brain collected at time of terminal euthanasia (NSGS mice: 2–3 DPI for Hi or 3–4 DPI for Lo; Hu-NSG-SGM3 mice: 2 DPI for Hi or 3 DPI for Lo) determined by qRT-PCR (mean ± SD). Blood was obtained from all but 3 mice (Lo-13-wk-1 [ID 5082–15], and one mouse each in the Hi- and Lo-NSGS groups). Significant at confidence of *p ≦ 0.05 (NSGS vs. Hu-NSG-SGM3 at same dose); **p

    Article Snippet: RNA was quantitated using a one-step real-time RT-PCR targeting the RVFV L gene sequence [ ], with values normalized to 18S transcript levels (SuperScript III Platinum One-Step qRT-PCR Kit, Thermo Fisher Scientific).

    Techniques: Mouse Assay, Quantitative RT-PCR