Structured Review

Roche pcr amplification
<t>PCR</t> results of <t>mecA</t> gene in 7 isolates staphylococcus epidermidis. Column 10: Size marker (50 bp). Column 8, 1-6: Isolates of Staphylococcus epidermidis containing mecA gene. Column 7: Positive control of staphylococcus aureus ATC 33591. Column 9: Negative control of staphylococcus aureus ATCC 25923
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1) Product Images from "Detection of methicillin-resistance gene in Staphylococcus epidermidis strains isolated from patients in Al-Zahra Hospital using polymerase chain reaction and minimum inhibitory concentration methods"

Article Title: Detection of methicillin-resistance gene in Staphylococcus epidermidis strains isolated from patients in Al-Zahra Hospital using polymerase chain reaction and minimum inhibitory concentration methods

Journal: Advanced Biomedical Research

doi: 10.4103/2277-9175.108008

PCR results of mecA gene in 7 isolates staphylococcus epidermidis. Column 10: Size marker (50 bp). Column 8, 1-6: Isolates of Staphylococcus epidermidis containing mecA gene. Column 7: Positive control of staphylococcus aureus ATC 33591. Column 9: Negative control of staphylococcus aureus ATCC 25923
Figure Legend Snippet: PCR results of mecA gene in 7 isolates staphylococcus epidermidis. Column 10: Size marker (50 bp). Column 8, 1-6: Isolates of Staphylococcus epidermidis containing mecA gene. Column 7: Positive control of staphylococcus aureus ATC 33591. Column 9: Negative control of staphylococcus aureus ATCC 25923

Techniques Used: Polymerase Chain Reaction, Marker, Positive Control, Negative Control

2) Product Images from "Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer"

Article Title: Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer

Journal: BMC Cancer

doi: 10.1186/1471-2407-13-526

DNA methylation and expression level of the PHD3 gene in HCT116 and DLD-1 CRC cells. A. HCT116 and DLD-1 cells were cultured under normoxic or hypoxic (1% O 2 ) conditions for 48 hrs. Cells were then used for DNA isolation followed by bisulfite modification. Methylation percentage of three DNA fragments within the PHD3 CpG island (Additional file 1 , Additional file 2 ) in HCT116 and DLD-1 cells under hypoxic and normoxic conditions was determined by Real Time PCR amplification of bisulfite treated standard and cell line DNA, followed by comparison of their HRM profiles. B. Cells were cultured in DMEM either in hypoxic (1%O 2 ) or normoxic conditions for 48 hrs. After incubation, the cells were used for total RNA isolation and reverse transcription. The PHD3 cDNA levels were determined by RQ-PCR relative quantification analysis. RQ-PCR results were standardized by the geometric mean of PBGD and hMRPL19 cDNA levels. PHD3 cDNA levels are expressed as a multiplicity of these cDNA copies in the cell line’s calibrator. C. Cells were cultured in DMEM either in hypoxic (1%O 2 ) ( H ) or normoxic ( N ) conditions for 48 hrs. Cells were then used for protein isolation. Proteins were separated by 10% SDS-PAGE, and transferred to a membrane that was then immunoblotted with Rp anti - PHD3 Ab and incubated with goat anti-rabbit HRP-conjugated Ab. The membrane was then stripped and reblotted with Rp anti-GAPDH Ab, followed by incubation with goat anti-rabbit HRP-conjugated Ab. The band densitometry readings were normalized to GAPDH loading control. The ratio of PHD3 to GAPDH for DLD-1 in normoxic conditions was assumed to be 1.
Figure Legend Snippet: DNA methylation and expression level of the PHD3 gene in HCT116 and DLD-1 CRC cells. A. HCT116 and DLD-1 cells were cultured under normoxic or hypoxic (1% O 2 ) conditions for 48 hrs. Cells were then used for DNA isolation followed by bisulfite modification. Methylation percentage of three DNA fragments within the PHD3 CpG island (Additional file 1 , Additional file 2 ) in HCT116 and DLD-1 cells under hypoxic and normoxic conditions was determined by Real Time PCR amplification of bisulfite treated standard and cell line DNA, followed by comparison of their HRM profiles. B. Cells were cultured in DMEM either in hypoxic (1%O 2 ) or normoxic conditions for 48 hrs. After incubation, the cells were used for total RNA isolation and reverse transcription. The PHD3 cDNA levels were determined by RQ-PCR relative quantification analysis. RQ-PCR results were standardized by the geometric mean of PBGD and hMRPL19 cDNA levels. PHD3 cDNA levels are expressed as a multiplicity of these cDNA copies in the cell line’s calibrator. C. Cells were cultured in DMEM either in hypoxic (1%O 2 ) ( H ) or normoxic ( N ) conditions for 48 hrs. Cells were then used for protein isolation. Proteins were separated by 10% SDS-PAGE, and transferred to a membrane that was then immunoblotted with Rp anti - PHD3 Ab and incubated with goat anti-rabbit HRP-conjugated Ab. The membrane was then stripped and reblotted with Rp anti-GAPDH Ab, followed by incubation with goat anti-rabbit HRP-conjugated Ab. The band densitometry readings were normalized to GAPDH loading control. The ratio of PHD3 to GAPDH for DLD-1 in normoxic conditions was assumed to be 1.

Techniques Used: DNA Methylation Assay, Expressing, Cell Culture, DNA Extraction, Modification, Methylation, Real-time Polymerase Chain Reaction, Amplification, Incubation, Isolation, Polymerase Chain Reaction, SDS Page

DNA methylation assessment of PHD3 gene regulatory region by bisulfite sequencing and HRM analysis in primary tissue samples from patients with CRC. Primary cancerous and histopathologically unchanged tissues from the same patients with CRC (P1-P5) were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. The PHD3 regions containing 60 CpG dinucleotides (chr14: 34 419 929-34 420 563) (Top panel A ) and 44 CpG dinucleotides (ch14: 34 419 346-34 419 943) (Top panel B ) were then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence (Additional file 1 , Additional file 2 ). The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from five positive bacterial clones was used for commercial sequencing. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and BDPC web server [ 23 , 24 ]. Black and grey boxes represent methylated and unmethylated CpG dinucleotide, respectively. Red rectangles correspond to regions amplified in HRM analysis by specific primers PHD3.1 (chr14: 34 419 922-34 420 080), PHD3.2 (chr14: 34 419 795- 34 419 935) and PHD3.3 (chr14: 34 419 400-34 419 538) (Additional file 1 , Additional file 2 ). Bottom panels A and B represent HRM profiles of standard and example of patient DNA (patient P2 from bisulfite sequencing) PCR product. Methylation percentage of three DNA fragments within the PHD3 CpG island was determined by Real Time PCR amplification of bisulfite treated standard and patient DNA, followed by comparison of their HRM profiles. DNA standards were prepared by mixing different ratios of methylated and non-methylated bisulfite treated DNA. HRM methylation analysis was performed using Light Cycler®480 Gene Scanning software, Roche Diagnostics GmbH (Mannheim, Germany). Each PCR amplification and HRM profile analysis was performed in triplicate.
Figure Legend Snippet: DNA methylation assessment of PHD3 gene regulatory region by bisulfite sequencing and HRM analysis in primary tissue samples from patients with CRC. Primary cancerous and histopathologically unchanged tissues from the same patients with CRC (P1-P5) were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. The PHD3 regions containing 60 CpG dinucleotides (chr14: 34 419 929-34 420 563) (Top panel A ) and 44 CpG dinucleotides (ch14: 34 419 346-34 419 943) (Top panel B ) were then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence (Additional file 1 , Additional file 2 ). The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from five positive bacterial clones was used for commercial sequencing. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and BDPC web server [ 23 , 24 ]. Black and grey boxes represent methylated and unmethylated CpG dinucleotide, respectively. Red rectangles correspond to regions amplified in HRM analysis by specific primers PHD3.1 (chr14: 34 419 922-34 420 080), PHD3.2 (chr14: 34 419 795- 34 419 935) and PHD3.3 (chr14: 34 419 400-34 419 538) (Additional file 1 , Additional file 2 ). Bottom panels A and B represent HRM profiles of standard and example of patient DNA (patient P2 from bisulfite sequencing) PCR product. Methylation percentage of three DNA fragments within the PHD3 CpG island was determined by Real Time PCR amplification of bisulfite treated standard and patient DNA, followed by comparison of their HRM profiles. DNA standards were prepared by mixing different ratios of methylated and non-methylated bisulfite treated DNA. HRM methylation analysis was performed using Light Cycler®480 Gene Scanning software, Roche Diagnostics GmbH (Mannheim, Germany). Each PCR amplification and HRM profile analysis was performed in triplicate.

Techniques Used: DNA Methylation Assay, Methylation Sequencing, DNA Extraction, Amplification, Modification, Sequencing, Polymerase Chain Reaction, Purification, Clone Assay, Plasmid Preparation, Isolation, Software, Methylation, Real-time Polymerase Chain Reaction

Ratio of cancerous PHD3 mRNA level to histopathologically unchanged tissue PHD3 mRNA level in three ranges of PHD3 methylation status: 0–1%; 1–10% and 10–100%. Methylation percentage of three DNA fragments within the PHD3 CpG island (Additional file 1 , Additional file 2 ) was determined by Real Time PCR amplification of bisulfite treated standard and patient DNA, followed by comparison of their HRM profiles. The methylation for each patient was calculated as an average percentage of methylation in amplified fragments located in the CpG island of PHD3. The samples were divided into three groups for statistical analysis: 0–1% methylation, 1–10% methylation and 10–100% methylation (Table 2 ) [ 28 - 30 ]. To evaluate the statistically significant difference in the ratio of cancerous PHD3 mRNA level to histopathologically unchanged tissue PHD3 mRNA level between the three DNA methylation ranges (0–1% methylation, 1–10% methylation and 10–100% methylation), the non-parametric Kruskal-Wallis test was employed.
Figure Legend Snippet: Ratio of cancerous PHD3 mRNA level to histopathologically unchanged tissue PHD3 mRNA level in three ranges of PHD3 methylation status: 0–1%; 1–10% and 10–100%. Methylation percentage of three DNA fragments within the PHD3 CpG island (Additional file 1 , Additional file 2 ) was determined by Real Time PCR amplification of bisulfite treated standard and patient DNA, followed by comparison of their HRM profiles. The methylation for each patient was calculated as an average percentage of methylation in amplified fragments located in the CpG island of PHD3. The samples were divided into three groups for statistical analysis: 0–1% methylation, 1–10% methylation and 10–100% methylation (Table 2 ) [ 28 - 30 ]. To evaluate the statistically significant difference in the ratio of cancerous PHD3 mRNA level to histopathologically unchanged tissue PHD3 mRNA level between the three DNA methylation ranges (0–1% methylation, 1–10% methylation and 10–100% methylation), the non-parametric Kruskal-Wallis test was employed.

Techniques Used: Methylation, Real-time Polymerase Chain Reaction, Amplification, DNA Methylation Assay

Effect of 5-dAzaC on PHD3 gene DNA methylation in HCT116 and DLD-1 CRC cells. HCT116 and DLD-1 cells were cultured under normoxic or hypoxic (1% O 2 ) conditions either in the absence or in the presence of 5-dAzaC at a concentration of 5.00 μM for 48 hrs. Cells were then used for DNA isolation followed by bisulfite modification. Methylation percentage of three DNA fragments within the PHD3 CpG island: A (chr14: 34 419 922–34 420 080), B (chr14: 34 419 795–34 419 935) and C (chr14: 34 419 400–34 419 538) (Additional file 1 , Additional file 2 ) in HCT116 and DLD-1 cells under hypoxic and normoxic conditions was determined by Real Time PCR amplification of bisulfite treated standard and cell line DNA, followed by comparison of their HRM profiles.
Figure Legend Snippet: Effect of 5-dAzaC on PHD3 gene DNA methylation in HCT116 and DLD-1 CRC cells. HCT116 and DLD-1 cells were cultured under normoxic or hypoxic (1% O 2 ) conditions either in the absence or in the presence of 5-dAzaC at a concentration of 5.00 μM for 48 hrs. Cells were then used for DNA isolation followed by bisulfite modification. Methylation percentage of three DNA fragments within the PHD3 CpG island: A (chr14: 34 419 922–34 420 080), B (chr14: 34 419 795–34 419 935) and C (chr14: 34 419 400–34 419 538) (Additional file 1 , Additional file 2 ) in HCT116 and DLD-1 cells under hypoxic and normoxic conditions was determined by Real Time PCR amplification of bisulfite treated standard and cell line DNA, followed by comparison of their HRM profiles.

Techniques Used: DNA Methylation Assay, Cell Culture, Concentration Assay, DNA Extraction, Modification, Methylation, Real-time Polymerase Chain Reaction, Amplification

3) Product Images from "Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates"

Article Title: Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates

Journal: Nucleic Acids Research

doi:

PCR product ratio (with/without exonuclease III) as a function of the percentage of heat-induced mtDNA loss. Four Qiagen-extracted mouse liver DNA samples were heated at 99°C for 0, 30, 60 and 90 s. Residual mtDNA was quantitated by Southern blot with an mtDNA probe while a 8636 bp mtDNA fragment was amplified with Protocol 1b, with or without 25 U of exonuclease III.
Figure Legend Snippet: PCR product ratio (with/without exonuclease III) as a function of the percentage of heat-induced mtDNA loss. Four Qiagen-extracted mouse liver DNA samples were heated at 99°C for 0, 30, 60 and 90 s. Residual mtDNA was quantitated by Southern blot with an mtDNA probe while a 8636 bp mtDNA fragment was amplified with Protocol 1b, with or without 25 U of exonuclease III.

Techniques Used: Polymerase Chain Reaction, Southern Blot, Amplification

Exonuclease III enhances long PCR amplification from phenol-extracted DNA samples. DNA samples were extracted with phenol/chloroform and either stored at –20 or –80°C for several years (mouse and human DNA, respectively) or used immediately (rat DNA). After PCR, agarose gels (0.7–1.2%) were loaded with 22 µl of the PCR products together with Hin dIII-digested phage λ DNA (M). ( A ) Five mouse liver DNA samples (ML1–ML5) were used for PCR co-amplification of the 316 and 8636 bp mtDNA fragments, using Protocol 1a without (exo 0) or with 25 U of exonuclease III (exo +). ( B ) Four rat liver DNA samples (RL1–RL4) were used for long PCR amplification of a 15.4 kb mtDNA fragment, using Protocol 2 without (exo 0) or with 25 U of exonuclease III (exo +). ( C ) Five human blood DNA samples (HB1–HB5) were used for long PCR amplification of a 5 kb fragment from the human CYP2D6 nuclear gene, using Protocol 3 without (exo 0) or with 50 U of exonuclease III (exo +).
Figure Legend Snippet: Exonuclease III enhances long PCR amplification from phenol-extracted DNA samples. DNA samples were extracted with phenol/chloroform and either stored at –20 or –80°C for several years (mouse and human DNA, respectively) or used immediately (rat DNA). After PCR, agarose gels (0.7–1.2%) were loaded with 22 µl of the PCR products together with Hin dIII-digested phage λ DNA (M). ( A ) Five mouse liver DNA samples (ML1–ML5) were used for PCR co-amplification of the 316 and 8636 bp mtDNA fragments, using Protocol 1a without (exo 0) or with 25 U of exonuclease III (exo +). ( B ) Four rat liver DNA samples (RL1–RL4) were used for long PCR amplification of a 15.4 kb mtDNA fragment, using Protocol 2 without (exo 0) or with 25 U of exonuclease III (exo +). ( C ) Five human blood DNA samples (HB1–HB5) were used for long PCR amplification of a 5 kb fragment from the human CYP2D6 nuclear gene, using Protocol 3 without (exo 0) or with 50 U of exonuclease III (exo +).

Techniques Used: Polymerase Chain Reaction, Amplification

Exonuclease III enhances long PCR amplification of the 8636 bp mtDNA fragment from depurinated mouse liver DNA samples. Aliquots of the same Qiagen-extracted mouse liver DNA preparation were treated in depurination buffer at 70°C for 0, 20, 40 or 60 min (AP0, AP20, AP40 and AP60, respectively) and the 8636 bp mtDNA fragment was amplified with Protocol 1b without (exo 0) or with 25 U of exonuclease III (exo +). The agarose gel (0.8%) was loaded with 22 µl of the PCR products. M, Hin dIII-digested phage λ DNA.
Figure Legend Snippet: Exonuclease III enhances long PCR amplification of the 8636 bp mtDNA fragment from depurinated mouse liver DNA samples. Aliquots of the same Qiagen-extracted mouse liver DNA preparation were treated in depurination buffer at 70°C for 0, 20, 40 or 60 min (AP0, AP20, AP40 and AP60, respectively) and the 8636 bp mtDNA fragment was amplified with Protocol 1b without (exo 0) or with 25 U of exonuclease III (exo +). The agarose gel (0.8%) was loaded with 22 µl of the PCR products. M, Hin dIII-digested phage λ DNA.

Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

Effect of exonuclease III on long PCR amplification performed with either rTth DNA polymerase alone (rTth) or in combination with Vent DNA polymerase (Vent). Aliquots of two phenol-extracted mouse liver DNA samples were used for long PCR amplification of the 8636 bp mtDNA fragment in the absence (exo 0) or presence of 25 U of exonuclease III (exo +). The 0.8% agarose gel was loaded with 22 µl of the PCR products. M, Hin dIII-digested phage λ DNA.
Figure Legend Snippet: Effect of exonuclease III on long PCR amplification performed with either rTth DNA polymerase alone (rTth) or in combination with Vent DNA polymerase (Vent). Aliquots of two phenol-extracted mouse liver DNA samples were used for long PCR amplification of the 8636 bp mtDNA fragment in the absence (exo 0) or presence of 25 U of exonuclease III (exo +). The 0.8% agarose gel was loaded with 22 µl of the PCR products. M, Hin dIII-digested phage λ DNA.

Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

Escherichia coli exonuclease III enhances long PCR amplification of mtDNA from heat-damaged mouse liver DNA templates. Qiagen-extracted mouse liver DNA was heated at 99°C for 30–120 s and two distinct regions of the mtDNA were co-amplified with Protocol 1a using 14 pmol of primers for the 316 bp PCR product and 40 pmol for the 8636 bp PCR product. Lanes 1–4 correspond to aliquots of the same mouse liver DNA sample heated for 30, 60, 90 and 120 s, respectively. Agarose gels (1.2%) were loaded with 22 µl of the products. M is Hin dIII-digested phage λ DNA (fragment sizes 23.1, 9.4, 6.6, 4.4, 2.3, 2.0 and 0.56 kb). ( A ) PCR reactions were performed without exonuclease III (exo 0) or with 25 U of exonuclease III (exo 25 U). ( B ) PCR reactions were performed with either 5 or 1 U of exonuclease III (exo 5 U and exo 1 U) or with 25 U of exonuclease III preheated at 99°C for 10 min (preheated exo).
Figure Legend Snippet: Escherichia coli exonuclease III enhances long PCR amplification of mtDNA from heat-damaged mouse liver DNA templates. Qiagen-extracted mouse liver DNA was heated at 99°C for 30–120 s and two distinct regions of the mtDNA were co-amplified with Protocol 1a using 14 pmol of primers for the 316 bp PCR product and 40 pmol for the 8636 bp PCR product. Lanes 1–4 correspond to aliquots of the same mouse liver DNA sample heated for 30, 60, 90 and 120 s, respectively. Agarose gels (1.2%) were loaded with 22 µl of the products. M is Hin dIII-digested phage λ DNA (fragment sizes 23.1, 9.4, 6.6, 4.4, 2.3, 2.0 and 0.56 kb). ( A ) PCR reactions were performed without exonuclease III (exo 0) or with 25 U of exonuclease III (exo 25 U). ( B ) PCR reactions were performed with either 5 or 1 U of exonuclease III (exo 5 U and exo 1 U) or with 25 U of exonuclease III preheated at 99°C for 10 min (preheated exo).

Techniques Used: Polymerase Chain Reaction, Amplification

4) Product Images from "Up-regulation of C5a receptor expression and function on human monocyte derived dendritic cells by prostaglandin E2"

Article Title: Up-regulation of C5a receptor expression and function on human monocyte derived dendritic cells by prostaglandin E2

Journal: Immunology

doi: 10.1111/j.1365-2567.2003.01764.x

Immature monocyte derived dendritic cells express the C5aR on the protein level (a) and mRNA level (b). (a) Binding of anti C5aR monoclonal antibody P12/1 to immature DC as determined by fluorescence cytometry. The histogram of the isotype control (thin line) and C5aR monoclonal antibody P12/1 binding (thick line) is shown. The specifity of binding was tested by preincubation of the antibody P12/1 with a 20fold weight excess of the peptide EX1 (dotted line), which had been used for generating the antibody, and by preincubation of the cells with C5a (dashed line). The preincubation prevented the binding of P12/1 to the C5aR. Anti-MHC class II antibody staining was not altered by preincubation of the antibody with EX1 peptide or preincubation of cells with C5a. (b). Detection of C5aR in immature DC by LightCycler RT-PCR. LightCycler melting curve analysis showed the specific peak for C5aR, which is clearly distinct from the peak caused by primer-dimer formation visible in the water (= negative) control. PBMC were used as positive control. Analysis of LightCycler PCR products by agarose gel electrophoresis revealed bands of the expected size (381 bp).
Figure Legend Snippet: Immature monocyte derived dendritic cells express the C5aR on the protein level (a) and mRNA level (b). (a) Binding of anti C5aR monoclonal antibody P12/1 to immature DC as determined by fluorescence cytometry. The histogram of the isotype control (thin line) and C5aR monoclonal antibody P12/1 binding (thick line) is shown. The specifity of binding was tested by preincubation of the antibody P12/1 with a 20fold weight excess of the peptide EX1 (dotted line), which had been used for generating the antibody, and by preincubation of the cells with C5a (dashed line). The preincubation prevented the binding of P12/1 to the C5aR. Anti-MHC class II antibody staining was not altered by preincubation of the antibody with EX1 peptide or preincubation of cells with C5a. (b). Detection of C5aR in immature DC by LightCycler RT-PCR. LightCycler melting curve analysis showed the specific peak for C5aR, which is clearly distinct from the peak caused by primer-dimer formation visible in the water (= negative) control. PBMC were used as positive control. Analysis of LightCycler PCR products by agarose gel electrophoresis revealed bands of the expected size (381 bp).

Techniques Used: Derivative Assay, Binding Assay, Fluorescence, Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Polymerase Chain Reaction, Agarose Gel Electrophoresis

DC(INF-γ) (matured with TNF-α+ IFN-γ) and DC(PGE 2 ) (matured with TNF-α+ PGE 2 show significant differences in C5aR expression, while CD83 is up-regulated on both DC(INF-γ) and DC(PGE 2 ). One representative experiment is shown in (a) (numbers are percentage of cells in the corresponding quadrants), and the mean ± SEM of seven independent experiments is shown in (b). (c) The difference of C5aR expression on DC(INF-γ) and DC(PGE 2 ) on the mRNA level by LightCycler real time PCR.
Figure Legend Snippet: DC(INF-γ) (matured with TNF-α+ IFN-γ) and DC(PGE 2 ) (matured with TNF-α+ PGE 2 show significant differences in C5aR expression, while CD83 is up-regulated on both DC(INF-γ) and DC(PGE 2 ). One representative experiment is shown in (a) (numbers are percentage of cells in the corresponding quadrants), and the mean ± SEM of seven independent experiments is shown in (b). (c) The difference of C5aR expression on DC(INF-γ) and DC(PGE 2 ) on the mRNA level by LightCycler real time PCR.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

5) Product Images from "Expression of HOXA11 in the mid-luteal endometrium from women with endometriosis-associated infertility"

Article Title: Expression of HOXA11 in the mid-luteal endometrium from women with endometriosis-associated infertility

Journal: Reproductive Biology and Endocrinology : RB & E

doi: 10.1186/1477-7827-10-1

Percentage of methylation of HOXA11 , region II in infertile women with endometriosis (E1-18), fertile women (C1-16) and infertile women with tubal occlusion (S1-S16) . Eutopic mid-luteal endometrium samples were used for genomic DNA isolation followed by bisulfite conversion of cytosine bases to uracil. The HOXA11 II region was then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence (Aditional file 1, Table S1; Aditional file 2, Figure S1). The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from ten positive bacterial clones was used for commercial sequencing. The results of bisulphite sequencing were assessed and presented using BiQ analyzer software [ 31 ] and BDPC web server [ 32 ] a Mann-Whitney Rank Sum Test.
Figure Legend Snippet: Percentage of methylation of HOXA11 , region II in infertile women with endometriosis (E1-18), fertile women (C1-16) and infertile women with tubal occlusion (S1-S16) . Eutopic mid-luteal endometrium samples were used for genomic DNA isolation followed by bisulfite conversion of cytosine bases to uracil. The HOXA11 II region was then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence (Aditional file 1, Table S1; Aditional file 2, Figure S1). The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from ten positive bacterial clones was used for commercial sequencing. The results of bisulphite sequencing were assessed and presented using BiQ analyzer software [ 31 ] and BDPC web server [ 32 ] a Mann-Whitney Rank Sum Test.

Techniques Used: Methylation, DNA Extraction, Amplification, Modification, Sequencing, Polymerase Chain Reaction, Purification, Clone Assay, Plasmid Preparation, Isolation, Bisulfite Sequencing, Software, MANN-WHITNEY

6) Product Images from "A DNA Vaccine against Yellow Fever Virus: Development and Evaluation"

Article Title: A DNA Vaccine against Yellow Fever Virus: Development and Evaluation

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0003693

p/YFE and pL/YFE DNA vaccine construct design. The fragment pM/M-E (extending from nucleotides 392 to 2452, black arrows) was amplified using PCR and cloned into a p43.2 vector to generate the p/YFE vector. This construct starts with the ER capsid signal and ends with the envelope trans-membrane domain. To generate the pL/YFE construct, we designed a second forward primer that annealed just upstream of the envelope trans-membrane domain to amplify the fragment extending from nucleotides 392 to 2323. This fragment was fused to the C-terminal end of LAMP and cloned into the same vector.
Figure Legend Snippet: p/YFE and pL/YFE DNA vaccine construct design. The fragment pM/M-E (extending from nucleotides 392 to 2452, black arrows) was amplified using PCR and cloned into a p43.2 vector to generate the p/YFE vector. This construct starts with the ER capsid signal and ends with the envelope trans-membrane domain. To generate the pL/YFE construct, we designed a second forward primer that annealed just upstream of the envelope trans-membrane domain to amplify the fragment extending from nucleotides 392 to 2323. This fragment was fused to the C-terminal end of LAMP and cloned into the same vector.

Techniques Used: Construct, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation

7) Product Images from "The NADPH oxidase Nox4 restricts the replicative lifespan of human endothelial cells"

Article Title: The NADPH oxidase Nox4 restricts the replicative lifespan of human endothelial cells

Journal: Biochemical Journal

doi: 10.1042/BJ20090666

Nox4 expression and activity in young and senescent HUVECs ( A ) HUVECs from four different donors were grown to senescence. RNA was prepared at early (y) and late (sen) passages and analysed for expression of Nox4 by RT–PCR (upper panel) and qPCR (lower panel). Results are means±S.D. for three independent experiments. ( B ) Cells of HUVEC strain #1 were analysed for ROS production by measurement of luminol-enhanced chemiluminescence at early passage (passage 7) and at senescence (passage 25), as indicated. At 20 min after onset of the experiment, DPI (10 μM) was added to specifically inhibit Nox activity. Results are means±S.D. for three independent experiments.
Figure Legend Snippet: Nox4 expression and activity in young and senescent HUVECs ( A ) HUVECs from four different donors were grown to senescence. RNA was prepared at early (y) and late (sen) passages and analysed for expression of Nox4 by RT–PCR (upper panel) and qPCR (lower panel). Results are means±S.D. for three independent experiments. ( B ) Cells of HUVEC strain #1 were analysed for ROS production by measurement of luminol-enhanced chemiluminescence at early passage (passage 7) and at senescence (passage 25), as indicated. At 20 min after onset of the experiment, DPI (10 μM) was added to specifically inhibit Nox activity. Results are means±S.D. for three independent experiments.

Techniques Used: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

8) Product Images from "Nodulin 41, a novel late nodulin of common bean with peptidase activity"

Article Title: Nodulin 41, a novel late nodulin of common bean with peptidase activity

Journal: BMC Plant Biology

doi: 10.1186/1471-2229-11-134

PvNod41 primary sequence . PvNod41 gene sequence (lower case) and protein sequence (upper case). PvNod41 encodes a 437 amino acid single polypeptide containing Asp-Thr-Gly and Asp-Ser-Gly sequences (DTG and DSG). Conserved motifs around the two catalytic aspartic acid residues are shown in boldface and underlined. Primer sequences used for PCR amplification are underlined. The arrow indicates the cleavage position of the putative signal peptide that directs the protein to the ER. HPLC-purified peptide sequences obtained from the trypsin digestion of PvNod41 [N-terminal end (N-term) as well as three internal peptides (P-1, P-2 and P-3)] are also depicted in this figure. The stop codon is marked with an asterisk.
Figure Legend Snippet: PvNod41 primary sequence . PvNod41 gene sequence (lower case) and protein sequence (upper case). PvNod41 encodes a 437 amino acid single polypeptide containing Asp-Thr-Gly and Asp-Ser-Gly sequences (DTG and DSG). Conserved motifs around the two catalytic aspartic acid residues are shown in boldface and underlined. Primer sequences used for PCR amplification are underlined. The arrow indicates the cleavage position of the putative signal peptide that directs the protein to the ER. HPLC-purified peptide sequences obtained from the trypsin digestion of PvNod41 [N-terminal end (N-term) as well as three internal peptides (P-1, P-2 and P-3)] are also depicted in this figure. The stop codon is marked with an asterisk.

Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, High Performance Liquid Chromatography, Purification

9) Product Images from "Production of in vitro amplified DNA pseudolibraries and high-throughput cDNA target amplification"

Article Title: Production of in vitro amplified DNA pseudolibraries and high-throughput cDNA target amplification

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-7-31

Result summary of gel electrophoreses with PCR products . Pictures of the gels were taken after staining with ethidium bromide. They were visually inspected and bands with suitable DNA size were scored (cf. fig. 2). Species identifiers: Hs, Homo sapiens; Gg, Gallus gallus (chicken); Dm, Drsosophila melanogaster; At, Arabidopsis thaliana. Domain abbreviations: Hel, helicase; ATP, ATPase, dsrm, double-stranded RNA-binding motiv. Designations: Species identifier, protein name, short designation of target domain(s).
Figure Legend Snippet: Result summary of gel electrophoreses with PCR products . Pictures of the gels were taken after staining with ethidium bromide. They were visually inspected and bands with suitable DNA size were scored (cf. fig. 2). Species identifiers: Hs, Homo sapiens; Gg, Gallus gallus (chicken); Dm, Drsosophila melanogaster; At, Arabidopsis thaliana. Domain abbreviations: Hel, helicase; ATP, ATPase, dsrm, double-stranded RNA-binding motiv. Designations: Species identifier, protein name, short designation of target domain(s).

Techniques Used: Polymerase Chain Reaction, Staining, RNA Binding Assay

10) Product Images from "Infectivity of Deinbollia mosaic virus, a novel weed-infecting begomovirus in East Africa"

Article Title: Infectivity of Deinbollia mosaic virus, a novel weed-infecting begomovirus in East Africa

Journal: Archives of Virology

doi: 10.1007/s00705-017-3495-x

Detection of DMV by hybridization and PCR amplification. The presence of DMV was confirmed by the presence of a deep purple signal on the nitrocellulose membrane in dot and tissue blot hybridization assays and by the amplification of a 450-bp PCR product using the B4/F and B4/R primer pair. The detection signals shown are from a D. borbonica infected with wild-type virus (A I, and B 1), healthy D. borbonica (A II), mock-inoculated N. benthamiana (A III, and C a), N. benthamiana inoculated with DNA partial dimers (A IV, B 2, and B 3), and N. benthamiana inoculated with concatemeric DNAs (A V, B 4, B 5, and C b)
Figure Legend Snippet: Detection of DMV by hybridization and PCR amplification. The presence of DMV was confirmed by the presence of a deep purple signal on the nitrocellulose membrane in dot and tissue blot hybridization assays and by the amplification of a 450-bp PCR product using the B4/F and B4/R primer pair. The detection signals shown are from a D. borbonica infected with wild-type virus (A I, and B 1), healthy D. borbonica (A II), mock-inoculated N. benthamiana (A III, and C a), N. benthamiana inoculated with DNA partial dimers (A IV, B 2, and B 3), and N. benthamiana inoculated with concatemeric DNAs (A V, B 4, B 5, and C b)

Techniques Used: Hybridization, Polymerase Chain Reaction, Amplification, Infection

11) Product Images from "RNAi combining Sleeping Beauty transposon system inhibits ex vivo expression of foot-and-mouth disease virus VP1 in transgenic sheep cells"

Article Title: RNAi combining Sleeping Beauty transposon system inhibits ex vivo expression of foot-and-mouth disease virus VP1 in transgenic sheep cells

Journal: Scientific Reports

doi: 10.1038/s41598-017-09302-1

Inhibitory effects of VP1 -shRNA on FMDV- VP1 . ( A ) Schematic map of the pLL3.7- VP1 -shRNA expression vector. ( B ) The VP1 gene amplification using overlapping PCR; 1: a-h, 2: a-g, 3: a-f, 4: a-e, 5: a-d, 6: a-c, 7: a-b, and M: marker. ( C ) Use of Dual-Glo luciferase to detect the inhibitory effect of targeted genes. 239FT cells were co-transfected with pll3.7-shRNA and psiCheck2 genes with different ratios, and the expression of Dual-Glo luciferase reporter genes was measured after 48 h. Data were expressed as the means ± S.E.M. (n = 3). Columns with different superscripts differ significantly, P
Figure Legend Snippet: Inhibitory effects of VP1 -shRNA on FMDV- VP1 . ( A ) Schematic map of the pLL3.7- VP1 -shRNA expression vector. ( B ) The VP1 gene amplification using overlapping PCR; 1: a-h, 2: a-g, 3: a-f, 4: a-e, 5: a-d, 6: a-c, 7: a-b, and M: marker. ( C ) Use of Dual-Glo luciferase to detect the inhibitory effect of targeted genes. 239FT cells were co-transfected with pll3.7-shRNA and psiCheck2 genes with different ratios, and the expression of Dual-Glo luciferase reporter genes was measured after 48 h. Data were expressed as the means ± S.E.M. (n = 3). Columns with different superscripts differ significantly, P

Techniques Used: shRNA, Expressing, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Marker, Luciferase, Transfection

Identification of SB transposon-mediated transgenic sheep. ( A ) Schematic diagram of the inserted part of the pUC- VP1 -shRNA expression vector, which was cut from pLL3.7 and ligated into the PUC19-IR/DR vector. ( B ) Pronuclear microinjection. (a): non-centrifuged fertilized ovine egg and (b): centrifuged fertilized egg (12000 × g for 5 min). ( C ) Electrophoresis of PCR products and ( D ) Southern blot analysis to identify the transgenic sheep, respectively. a: U6- VP1 -shRNA group and linearized pLL3.7- VP1 -shRNA plasmid group. b: IR/DR-U6- VP1 -shRNA + SB100 × group. WT = wild type as a negative control; “p”: positive control; “1–8”: transgenic lambs. ( E ) Inhibitory effects of shRNA on VP1 gene expression in ear fibroblasts of transgenic versus wild type sheep as determined by luciferase reporter assay. Each test was repeated three times for each individual. Tg ( = transgenic sheep), N = 8; WT (wild type), N = 8. ( F ) A photo of the transgenic lamb. Data were expressed as the means ± S.E.M. Columns with different superscripts differed significantly, P
Figure Legend Snippet: Identification of SB transposon-mediated transgenic sheep. ( A ) Schematic diagram of the inserted part of the pUC- VP1 -shRNA expression vector, which was cut from pLL3.7 and ligated into the PUC19-IR/DR vector. ( B ) Pronuclear microinjection. (a): non-centrifuged fertilized ovine egg and (b): centrifuged fertilized egg (12000 × g for 5 min). ( C ) Electrophoresis of PCR products and ( D ) Southern blot analysis to identify the transgenic sheep, respectively. a: U6- VP1 -shRNA group and linearized pLL3.7- VP1 -shRNA plasmid group. b: IR/DR-U6- VP1 -shRNA + SB100 × group. WT = wild type as a negative control; “p”: positive control; “1–8”: transgenic lambs. ( E ) Inhibitory effects of shRNA on VP1 gene expression in ear fibroblasts of transgenic versus wild type sheep as determined by luciferase reporter assay. Each test was repeated three times for each individual. Tg ( = transgenic sheep), N = 8; WT (wild type), N = 8. ( F ) A photo of the transgenic lamb. Data were expressed as the means ± S.E.M. Columns with different superscripts differed significantly, P

Techniques Used: Transgenic Assay, shRNA, Expressing, Plasmid Preparation, Electrophoresis, Polymerase Chain Reaction, Southern Blot, Negative Control, Positive Control, Luciferase, Reporter Assay

12) Product Images from "Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA"

Article Title: Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000965

pUL29/28 specifically activates the MIE promoter irrespective of HCMV pUL38 expression. (A) pUL29/28 activates the MIEP. Upper left panel: U2OS cells that stably maintained the empty pLXSN vector (U2OS) or pLXSN expressing HCMV pUL38 (USOS-38) were transfected with 50 ng of pGL3-MIEP reporter plasmid and 10, 100, or 500 ng of pCGN empty vector or pCGN-pUL29/28 effector plasmid. Luciferase activity was assayed 48 h posttransfection using equal protein amounts within each lysate and normalized to luciferase activity from empty vector. Lower panels: The levels of pUL29/28HA and pUL38 expression were assayed by Western blot analysis using the same lysates and antibody to HA, pUL38 or tubulin. Right panel: UL29 and luciferase RNA expression was determined by qRT-PCR from U2OS as compared to U2OS-38 cells. (B) pUL29/28 exhibits promoter-specific effects. Luciferase assays were completed using 500 ng of pCGN or pCGN-pUL29/28 and MIEP, ISRE and NF-κB promoter reporter constructs. The relative luciferase activity was determined as described above. (C) NuRD is required for optimal expression of an MIEP reporter. Left panel: Disruption of the NuRD complex in U-2 OS cells. Short hairpin RNA (shRNA) sequences to a scrambled control, CHD4 or RBBP4 were delivered to U-2 OS cells using lentiviruses and expressing cells were isolated by puromycin resistance. Expression of CHD4 and RBBP4 was quantified by qRT-PCR. The data was normalized to GAPDH RNA levels and includes the percent reduction for each gene relative to control. Right panel: Luciferase assays were completed using either empty pCGN or pCGN-pUL29/28 and MIEP reporter. The relative luciferase activity was determined as described above and the data is derived from replicate experiments (**p
Figure Legend Snippet: pUL29/28 specifically activates the MIE promoter irrespective of HCMV pUL38 expression. (A) pUL29/28 activates the MIEP. Upper left panel: U2OS cells that stably maintained the empty pLXSN vector (U2OS) or pLXSN expressing HCMV pUL38 (USOS-38) were transfected with 50 ng of pGL3-MIEP reporter plasmid and 10, 100, or 500 ng of pCGN empty vector or pCGN-pUL29/28 effector plasmid. Luciferase activity was assayed 48 h posttransfection using equal protein amounts within each lysate and normalized to luciferase activity from empty vector. Lower panels: The levels of pUL29/28HA and pUL38 expression were assayed by Western blot analysis using the same lysates and antibody to HA, pUL38 or tubulin. Right panel: UL29 and luciferase RNA expression was determined by qRT-PCR from U2OS as compared to U2OS-38 cells. (B) pUL29/28 exhibits promoter-specific effects. Luciferase assays were completed using 500 ng of pCGN or pCGN-pUL29/28 and MIEP, ISRE and NF-κB promoter reporter constructs. The relative luciferase activity was determined as described above. (C) NuRD is required for optimal expression of an MIEP reporter. Left panel: Disruption of the NuRD complex in U-2 OS cells. Short hairpin RNA (shRNA) sequences to a scrambled control, CHD4 or RBBP4 were delivered to U-2 OS cells using lentiviruses and expressing cells were isolated by puromycin resistance. Expression of CHD4 and RBBP4 was quantified by qRT-PCR. The data was normalized to GAPDH RNA levels and includes the percent reduction for each gene relative to control. Right panel: Luciferase assays were completed using either empty pCGN or pCGN-pUL29/28 and MIEP reporter. The relative luciferase activity was determined as described above and the data is derived from replicate experiments (**p

Techniques Used: Expressing, Stable Transfection, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Western Blot, RNA Expression, Quantitative RT-PCR, Construct, shRNA, Isolation, Derivative Assay

13) Product Images from "Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution"

Article Title: Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

Journal: bioRxiv

doi: 10.1101/2020.05.06.078147

Metagenomic analysis of the 16S rRNA gene amplicons using MinION™ nanopore sequencing. a Workflow of 16S rRNA amplicon sequencing on the MinION™ platform. Sequencing libraries are generated by the four-primer PCR-based strategy, enabling simplified post-PCR adapter attachment. At the initial stage of PCR, the 16S rRNA gene is amplified with the inner primer pairs. The resulting PCR products are targeted for amplification with the outer primers to introduce the barcode and tag sequences at both ends, to which adapter molecules can be attached in a single-step reaction. b, c Taxonomic assignments of a mock community analyzed by MinION™ sequencing. The V1-V9 or V3-V4 region of the 16S rRNA gene was amplified from a pre-characterized mock community sample comprising ten bacterial species and sequenced on the MinION™ platform. Three thousand reads were randomly selected from the processed data set and aligned directly to the reference genome database of 5850 representative bacterial species. The pie charts represent taxonomic profiles at the (b) genus and (c) species levels. Slices corresponding to misclassified (assigned to bacteria not present in the mock community) or unclassified (not classified at the species level but placed in a higher taxonomic rank) reads are exploded. The relative abundance (%) of each taxon is shown.
Figure Legend Snippet: Metagenomic analysis of the 16S rRNA gene amplicons using MinION™ nanopore sequencing. a Workflow of 16S rRNA amplicon sequencing on the MinION™ platform. Sequencing libraries are generated by the four-primer PCR-based strategy, enabling simplified post-PCR adapter attachment. At the initial stage of PCR, the 16S rRNA gene is amplified with the inner primer pairs. The resulting PCR products are targeted for amplification with the outer primers to introduce the barcode and tag sequences at both ends, to which adapter molecules can be attached in a single-step reaction. b, c Taxonomic assignments of a mock community analyzed by MinION™ sequencing. The V1-V9 or V3-V4 region of the 16S rRNA gene was amplified from a pre-characterized mock community sample comprising ten bacterial species and sequenced on the MinION™ platform. Three thousand reads were randomly selected from the processed data set and aligned directly to the reference genome database of 5850 representative bacterial species. The pie charts represent taxonomic profiles at the (b) genus and (c) species levels. Slices corresponding to misclassified (assigned to bacteria not present in the mock community) or unclassified (not classified at the species level but placed in a higher taxonomic rank) reads are exploded. The relative abundance (%) of each taxon is shown.

Techniques Used: Nanopore Sequencing, Amplification, Sequencing, Generated, Polymerase Chain Reaction, Introduce

14) Product Images from "Development of a Highly Sensitive and Specific Method for Detection of Circulating Tumor Cells Harboring Somatic Mutations in Non-Small-Cell Lung Cancer Patients"

Article Title: Development of a Highly Sensitive and Specific Method for Detection of Circulating Tumor Cells Harboring Somatic Mutations in Non-Small-Cell Lung Cancer Patients

Journal: PLoS ONE

doi: 10.1371/journal.pone.0085350

Sensitivity of EGFR DelEx19 mutation detection by real-time polymerase chain reaction and melting curve analysis. A) EGFR DelEx19 mutation detection in serially diluted DNA (50 ng/reaction) from A431 cells ( EGFR -wild type control) and NCI-HCC-827 cells ( EGFR DelEx19 mutant control 1). Melting peaks indicative of EGFR -wild type DNA (right) and EGFR DelEx19 (left) can be clearly distinguished. Real-time PCR reactions were carried out without addition of locked nucleic acids (LNA) and in serial DNA dilutions of up to 1∶16. Water (H 2 O, bottom line) and and 50 ng of undiluted genomic DNA EGFR -wild type (A431) and EGFR -mutant cells (NCI-HCC-827) were included as controls (representative examples of duplicate reactions). B) Reactions were conducted as in (A) but with addition of LNA (6 pmol). Note suppression of the EGFR -wild type signal, which allowed discrimination of the EGFR DelEx19 mutation signal up to a dilution of 1∶1,024 (
Figure Legend Snippet: Sensitivity of EGFR DelEx19 mutation detection by real-time polymerase chain reaction and melting curve analysis. A) EGFR DelEx19 mutation detection in serially diluted DNA (50 ng/reaction) from A431 cells ( EGFR -wild type control) and NCI-HCC-827 cells ( EGFR DelEx19 mutant control 1). Melting peaks indicative of EGFR -wild type DNA (right) and EGFR DelEx19 (left) can be clearly distinguished. Real-time PCR reactions were carried out without addition of locked nucleic acids (LNA) and in serial DNA dilutions of up to 1∶16. Water (H 2 O, bottom line) and and 50 ng of undiluted genomic DNA EGFR -wild type (A431) and EGFR -mutant cells (NCI-HCC-827) were included as controls (representative examples of duplicate reactions). B) Reactions were conducted as in (A) but with addition of LNA (6 pmol). Note suppression of the EGFR -wild type signal, which allowed discrimination of the EGFR DelEx19 mutation signal up to a dilution of 1∶1,024 (

Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction

15) Product Images from "A Non-coding RNA of Insect HzNV-1 Virus Establishes Latent Viral Infection through MicroRNA"

Article Title: A Non-coding RNA of Insect HzNV-1 Virus Establishes Latent Viral Infection through MicroRNA

Journal: Scientific Reports

doi: 10.1038/srep00060

Cloning and analysis of the predicted miRNA by stem-loop PCR and northern blot. Stem-loop PCR was performed to clone and analyze the proper expression of the predicted miRNAs in (A) Hz NV-1 productively infected cells, (B) pag1 -transfected cells, and (C) Hz NV-1 latently infected cells. (D, E) Predicted secondary structures of hv-miR-246 5P (D), and hv-miR-2959 5p (E), precursors. (F) Small RNAs harvested from Hz NV-1 productively infected cells at various time points were analysis by northern blots with probes against predicted Hz NV-1 miRNAs (top panels) or let-7a miRNA as a positive control (bottom panels).
Figure Legend Snippet: Cloning and analysis of the predicted miRNA by stem-loop PCR and northern blot. Stem-loop PCR was performed to clone and analyze the proper expression of the predicted miRNAs in (A) Hz NV-1 productively infected cells, (B) pag1 -transfected cells, and (C) Hz NV-1 latently infected cells. (D, E) Predicted secondary structures of hv-miR-246 5P (D), and hv-miR-2959 5p (E), precursors. (F) Small RNAs harvested from Hz NV-1 productively infected cells at various time points were analysis by northern blots with probes against predicted Hz NV-1 miRNAs (top panels) or let-7a miRNA as a positive control (bottom panels).

Techniques Used: Clone Assay, Polymerase Chain Reaction, Northern Blot, Expressing, Infection, Transfection, Positive Control

Establishment of latent viral infection by miRNAs. (A) Sf21 cells were transfected with or without miRNA followed by Hz NV-1 infection. (B) Column representation of the results of panel (A). (C) Confirmation of hhi1 and pag1 expressions in various cells by RT-PCR.
Figure Legend Snippet: Establishment of latent viral infection by miRNAs. (A) Sf21 cells were transfected with or without miRNA followed by Hz NV-1 infection. (B) Column representation of the results of panel (A). (C) Confirmation of hhi1 and pag1 expressions in various cells by RT-PCR.

Techniques Used: Infection, Transfection, Reverse Transcription Polymerase Chain Reaction

Down-regulation of hhi1 expression by pag1 , hv-miR-246, and hv-miR-2959. (A, C) sequences of hv-miR-2959 and hv-miR-246 were shown, and miRNAs with mutations were denoted as hv-miR-246m and hv-miR-2959m, separately. (B, D) levels of hhi1 transcript in various treatments were analyzed by northern hybridization (left panel) and RT-PCR (right panel).
Figure Legend Snippet: Down-regulation of hhi1 expression by pag1 , hv-miR-246, and hv-miR-2959. (A, C) sequences of hv-miR-2959 and hv-miR-246 were shown, and miRNAs with mutations were denoted as hv-miR-246m and hv-miR-2959m, separately. (B, D) levels of hhi1 transcript in various treatments were analyzed by northern hybridization (left panel) and RT-PCR (right panel).

Techniques Used: Expressing, Northern Blot, Hybridization, Reverse Transcription Polymerase Chain Reaction

Suppression of Hz NV-1 viral latency by knocking down pag1 expression. (A) RT-PCR showed that artificial siRNA can efficiently suppress pag1 expression in Hz NV-1 infected cells. (B) pag1 expression is not detectable by RT-PCR in the pag1 -null Hz NV-1-infected cells. (C) Formation of latent colony is not observed by the infection of pag1 -null Hz NV-1.
Figure Legend Snippet: Suppression of Hz NV-1 viral latency by knocking down pag1 expression. (A) RT-PCR showed that artificial siRNA can efficiently suppress pag1 expression in Hz NV-1 infected cells. (B) pag1 expression is not detectable by RT-PCR in the pag1 -null Hz NV-1-infected cells. (C) Formation of latent colony is not observed by the infection of pag1 -null Hz NV-1.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Infection

16) Product Images from "Vif Counteracts a Cyclophilin A-Imposed Inhibition of Simian Immunodeficiency Viruses in Human Cells ▿"

Article Title: Vif Counteracts a Cyclophilin A-Imposed Inhibition of Simian Immunodeficiency Viruses in Human Cells ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02727-06

Target cell TRIM5α does not affect infection by SIVagm. (A) Jurkat cells were transduced with an HIV-1-based vector that confers puromycin resistance and delivers an shRNA expression construct specific either for human TRIM5α (TR5-shRNA) or for luciferase (Luc-shRNA). VSV-G pseudotyped N- or B-tropic MLVGFP virions were normalized for titer with nonrestrictive Mus dunni cells and then used to infect Jurkat Luc-shRNA cells or Jurkat TR5-shRNA cells. The percentage of GFP-positive (infected) cells was determined by flow cytometry. Shown are representative results of a single experiment. Identical results were obtained on three separate occasions using independently produced viral stocks. (B) WT and vif -deficient (Vif−) SIVagm stocks were produced with untreated Jurkat cells and used to infect untreated Jurkat cells (normal), Jurkat TR5-shRNA cells (TRIM5α-KD), or Jurkat TR5-shRNA cells pretreated for 24 h with 2.5 μM CsA (CsA-treated TRIM5α-KD). Total DNA was harvested 24 h postinfection. Accumulation of full-length viral cDNA was determined by DNA PCR amplification. A primer set for the amplification of actin DNA was included in each reaction as an internal control (Actin). Heat, heat-inactivated WT SIVagm. (C) LuSIV cells were transduced with TR5-shRNA or Luc-shRNA vectors as described in the legend to panel A. TRIM5α silencing was measured by determining the relative sensitivity of the cells to infection by VSV-G pseudotyped B-tropic or N-tropic MLVGFP virions. (D) LuSIV TR5-shRNA cells were infected with equal amounts of the WT or the vif -defective [Vif(−)] SIVagm derived from infected Jurkat cells. Mock-infected cells were analyzed in parallel (mock). Infected cells were harvested 24 h after infection, and virus-induced luciferase activity was measured as described in Materials and Methods. Error bars reflect standard deviations calculated from three independent experiments.
Figure Legend Snippet: Target cell TRIM5α does not affect infection by SIVagm. (A) Jurkat cells were transduced with an HIV-1-based vector that confers puromycin resistance and delivers an shRNA expression construct specific either for human TRIM5α (TR5-shRNA) or for luciferase (Luc-shRNA). VSV-G pseudotyped N- or B-tropic MLVGFP virions were normalized for titer with nonrestrictive Mus dunni cells and then used to infect Jurkat Luc-shRNA cells or Jurkat TR5-shRNA cells. The percentage of GFP-positive (infected) cells was determined by flow cytometry. Shown are representative results of a single experiment. Identical results were obtained on three separate occasions using independently produced viral stocks. (B) WT and vif -deficient (Vif−) SIVagm stocks were produced with untreated Jurkat cells and used to infect untreated Jurkat cells (normal), Jurkat TR5-shRNA cells (TRIM5α-KD), or Jurkat TR5-shRNA cells pretreated for 24 h with 2.5 μM CsA (CsA-treated TRIM5α-KD). Total DNA was harvested 24 h postinfection. Accumulation of full-length viral cDNA was determined by DNA PCR amplification. A primer set for the amplification of actin DNA was included in each reaction as an internal control (Actin). Heat, heat-inactivated WT SIVagm. (C) LuSIV cells were transduced with TR5-shRNA or Luc-shRNA vectors as described in the legend to panel A. TRIM5α silencing was measured by determining the relative sensitivity of the cells to infection by VSV-G pseudotyped B-tropic or N-tropic MLVGFP virions. (D) LuSIV TR5-shRNA cells were infected with equal amounts of the WT or the vif -defective [Vif(−)] SIVagm derived from infected Jurkat cells. Mock-infected cells were analyzed in parallel (mock). Infected cells were harvested 24 h after infection, and virus-induced luciferase activity was measured as described in Materials and Methods. Error bars reflect standard deviations calculated from three independent experiments.

Techniques Used: Infection, Transduction, Plasmid Preparation, shRNA, Expressing, Construct, Luciferase, Flow Cytometry, Cytometry, Produced, Polymerase Chain Reaction, Amplification, Derivative Assay, Activity Assay

17) Product Images from "Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells"

Article Title: Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005872

The gene structure of the piggyBac transformation vector, pBac[pAAPP-mBax; 3xP3-EGFP], TG mosquito lines, and insertion sites. (A) The gene construct derived from the piggyBac -based vector contains piggyBac Left-arm (L) and Right-arm (R) with an inverted terminal repeat (ITR). The T7-mBax gene is expressed under the control of the An . stephensi aapp promoter (pAAPP) and An . gambiae trypsin terminator (Tryter). The transformation marker, EGFP is expressed under the control of the 3xP3 promoter. A double line represents the probe region for a Southern blot analysis. The restriction enzyme ( Msp I) site is represented below the scheme. The red arrow represents the primer sites for a RT-PCR analysis in Fig 2 . (B) A Southern blot analysis of AAPP-mBax lines. Genomic DNA from AAPP-mBax mosquito lines (lines 1 and 3) was digested with Msp I, and hybridized with a fragment corresponding to the piggyBac R region. (C) Insertion sites of the transgene in AAPP-mBax lines 1 and 3. The blue bars show the local DNA region within each genomic scaffold. Black boxes represent the annotated protein-coding region in the VectorBase ( https://www.vectorbase.org/ ). Double-headed arrows show the piggyBac construct. L: piggyBac Left-arm, R: piggyBac Right-arm.
Figure Legend Snippet: The gene structure of the piggyBac transformation vector, pBac[pAAPP-mBax; 3xP3-EGFP], TG mosquito lines, and insertion sites. (A) The gene construct derived from the piggyBac -based vector contains piggyBac Left-arm (L) and Right-arm (R) with an inverted terminal repeat (ITR). The T7-mBax gene is expressed under the control of the An . stephensi aapp promoter (pAAPP) and An . gambiae trypsin terminator (Tryter). The transformation marker, EGFP is expressed under the control of the 3xP3 promoter. A double line represents the probe region for a Southern blot analysis. The restriction enzyme ( Msp I) site is represented below the scheme. The red arrow represents the primer sites for a RT-PCR analysis in Fig 2 . (B) A Southern blot analysis of AAPP-mBax lines. Genomic DNA from AAPP-mBax mosquito lines (lines 1 and 3) was digested with Msp I, and hybridized with a fragment corresponding to the piggyBac R region. (C) Insertion sites of the transgene in AAPP-mBax lines 1 and 3. The blue bars show the local DNA region within each genomic scaffold. Black boxes represent the annotated protein-coding region in the VectorBase ( https://www.vectorbase.org/ ). Double-headed arrows show the piggyBac construct. L: piggyBac Left-arm, R: piggyBac Right-arm.

Techniques Used: Transformation Assay, Plasmid Preparation, Construct, Derivative Assay, Marker, Southern Blot, Reverse Transcription Polymerase Chain Reaction

Comparison of the P . berghei load in the midgut of mosquitoes. The expression of the P . berghei Pb47 gene in wild-type (WT) and AAPP-mBax (line 1) mosquitoes after blood feeding was analyzed by quantitative RT-PCR. Relative expression levels are shown, with the average value of wild-type mosquitoes being 1. The expression levels of Pb47 were normalized using that of the An . stephensi GAPDH gene. Two independent experiments were performed. No significant differences were observed between AAPP-mBax and wild-type mosquitoes. n.s., not significant ( P = 0.9377 in Exp 1 and P = 0.9672 in Exp 2).
Figure Legend Snippet: Comparison of the P . berghei load in the midgut of mosquitoes. The expression of the P . berghei Pb47 gene in wild-type (WT) and AAPP-mBax (line 1) mosquitoes after blood feeding was analyzed by quantitative RT-PCR. Relative expression levels are shown, with the average value of wild-type mosquitoes being 1. The expression levels of Pb47 were normalized using that of the An . stephensi GAPDH gene. Two independent experiments were performed. No significant differences were observed between AAPP-mBax and wild-type mosquitoes. n.s., not significant ( P = 0.9377 in Exp 1 and P = 0.9672 in Exp 2).

Techniques Used: Expressing, Quantitative RT-PCR

18) Product Images from "Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance"

Article Title: Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance

Journal: PLoS ONE

doi: 10.1371/journal.pone.0036494

Sequence coverage of three amplicons from a clonal HXB2 viral stock and HXB2 plasmid. The number of sequences from the clonal viral stock (red) or HIV plasmid (blue and black) that aligned to each nucleotide position of the NC_001802 HXB2 HIV reference sequence is shown across the pol gene. The HXB2 vRNA clonal viral stock was sequenced under one GS Junior sequencing run, while two independent PCR amplifications were used to sequence the plasmid under two different MID tags in a separate GS Junior sequencing run. Pyrosequencing was performed on three overlapping amplicons with nucleotide positions for each amplicon represented at the top of the graph.
Figure Legend Snippet: Sequence coverage of three amplicons from a clonal HXB2 viral stock and HXB2 plasmid. The number of sequences from the clonal viral stock (red) or HIV plasmid (blue and black) that aligned to each nucleotide position of the NC_001802 HXB2 HIV reference sequence is shown across the pol gene. The HXB2 vRNA clonal viral stock was sequenced under one GS Junior sequencing run, while two independent PCR amplifications were used to sequence the plasmid under two different MID tags in a separate GS Junior sequencing run. Pyrosequencing was performed on three overlapping amplicons with nucleotide positions for each amplicon represented at the top of the graph.

Techniques Used: Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Amplification

19) Product Images from "Time-series metagenomic analysis reveals robustness of soil microbiome against chemical disturbance"

Article Title: Time-series metagenomic analysis reveals robustness of soil microbiome against chemical disturbance

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

doi: 10.1093/dnares/dsv023

Phylum- and genus-level taxonomical succession of microbial communities in control and polluted soils. PCR amplicons of V3–V4 regions in the 16S rRNA genes from metagenomic samples were pyrosequenced, and taxonomically assigned by the RDP classifier (see the text for details). (A) Phylum-level succession. (B) Genus-level succession. C and M: metagenomic samples from the control and polluted soils, respectively. The numerals before C and M are the weeks after the pollution. Only the top 15 prokaryotic genera are shown for simplicity. Taxa with asterisks are genera incertae sedis .
Figure Legend Snippet: Phylum- and genus-level taxonomical succession of microbial communities in control and polluted soils. PCR amplicons of V3–V4 regions in the 16S rRNA genes from metagenomic samples were pyrosequenced, and taxonomically assigned by the RDP classifier (see the text for details). (A) Phylum-level succession. (B) Genus-level succession. C and M: metagenomic samples from the control and polluted soils, respectively. The numerals before C and M are the weeks after the pollution. Only the top 15 prokaryotic genera are shown for simplicity. Taxa with asterisks are genera incertae sedis .

Techniques Used: Polymerase Chain Reaction

20) Product Images from "Clinical significance of DNA methylation mRNA levels of TET family members in colorectal cancer"

Article Title: Clinical significance of DNA methylation mRNA levels of TET family members in colorectal cancer

Journal: Journal of Cancer Research and Clinical Oncology

doi: 10.1007/s00432-014-1901-2

DNA methylation assessment of TET1 , TET2 and TET3 gene regulatory region by bisulfite sequencing in tissue samples from patients with CRC. Primary cancerous and histopathologically unchanged tissues from the same patients with CRC (P1–P5) were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. The TET1, TET2 and TET3 regions containing 47, 64 and 40 CpG dinucleotides, respectively, were then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence. The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from five positive bacterial clones was used for commercial sequencing. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and BDPC web server. Black , gray and white boxes represent methylated, unmethylated or undetermined CpG dinucleotide, respectively
Figure Legend Snippet: DNA methylation assessment of TET1 , TET2 and TET3 gene regulatory region by bisulfite sequencing in tissue samples from patients with CRC. Primary cancerous and histopathologically unchanged tissues from the same patients with CRC (P1–P5) were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. The TET1, TET2 and TET3 regions containing 47, 64 and 40 CpG dinucleotides, respectively, were then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence. The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from five positive bacterial clones was used for commercial sequencing. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and BDPC web server. Black , gray and white boxes represent methylated, unmethylated or undetermined CpG dinucleotide, respectively

Techniques Used: DNA Methylation Assay, Methylation Sequencing, DNA Extraction, Amplification, Modification, Sequencing, Polymerase Chain Reaction, Purification, Clone Assay, Plasmid Preparation, Isolation, Software, Methylation

DNA methylation effect on TET1 mRNA levels in cancerous tissue. The primary cancerous tissues from 113 patients with CRC were used for RNA isolation. Total RNA was reverse-transcribed, and cDNAs were investigated by RQ-PCR relative quantification analysis. The TET1 mRNA levels were corrected by the geometric mean of PBGD and hMRPL19 cDNA levels. The amount of TET1 mRNA was expressed as the decimal logarithm of multiples of cDNA copies in the calibrator
Figure Legend Snippet: DNA methylation effect on TET1 mRNA levels in cancerous tissue. The primary cancerous tissues from 113 patients with CRC were used for RNA isolation. Total RNA was reverse-transcribed, and cDNAs were investigated by RQ-PCR relative quantification analysis. The TET1 mRNA levels were corrected by the geometric mean of PBGD and hMRPL19 cDNA levels. The amount of TET1 mRNA was expressed as the decimal logarithm of multiples of cDNA copies in the calibrator

Techniques Used: DNA Methylation Assay, Isolation, Polymerase Chain Reaction

DNA methylation assessment of TET1 , TET2 and TET3 gene regulatory region by HRM analysis in tissue samples from patients with CRC. Primary cancerous and histopathologically unchanged tissues from the same patients with CRC were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. a – c Represent HRM profiles of standard and example of patient DNA PCR product for TET1, TET2 and TET3, respectively. Methylation percentage of DNA fragments within the CpG island was determined by real-time PCR amplification of bisulfite-treated standard and patient DNA, followed by comparison of their HRM profiles. DNA standards were prepared by mixing different ratios of methylated and non-methylated bisulfite-treated standard DNA. HRM methylation analysis was performed using Light Cycler ® 480 or LightCycler ® 96 Gene Scanning software, Roche Diagnostics GmbH (Mannheim, Germany). Each PCR amplification and HRM profile analysis was performed in triplicate
Figure Legend Snippet: DNA methylation assessment of TET1 , TET2 and TET3 gene regulatory region by HRM analysis in tissue samples from patients with CRC. Primary cancerous and histopathologically unchanged tissues from the same patients with CRC were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. a – c Represent HRM profiles of standard and example of patient DNA PCR product for TET1, TET2 and TET3, respectively. Methylation percentage of DNA fragments within the CpG island was determined by real-time PCR amplification of bisulfite-treated standard and patient DNA, followed by comparison of their HRM profiles. DNA standards were prepared by mixing different ratios of methylated and non-methylated bisulfite-treated standard DNA. HRM methylation analysis was performed using Light Cycler ® 480 or LightCycler ® 96 Gene Scanning software, Roche Diagnostics GmbH (Mannheim, Germany). Each PCR amplification and HRM profile analysis was performed in triplicate

Techniques Used: DNA Methylation Assay, DNA Extraction, Polymerase Chain Reaction, Methylation, Real-time Polymerase Chain Reaction, Amplification, Software

21) Product Images from "Characterization of a eukaryotic translation initiation factor 5A homolog from Tamarix androssowii involved in plant abiotic stress tolerance"

Article Title: Characterization of a eukaryotic translation initiation factor 5A homolog from Tamarix androssowii involved in plant abiotic stress tolerance

Journal: BMC Plant Biology

doi: 10.1186/1471-2229-12-118

Analysis of TaeIF5A1 expression in response to different abiotic stresses. A–E. Two-month-old seedlings were watered with one of the following solutions containing 0.4 M NaCl, 20% PEG6000, 0.3 M NaHCO 3 , 150 μM CdCl 2 , or 150 μM ABA for the indicated times. The relative expression level = log2 (transcription level under stress treatment / transcription level under control conditions). The error bars were obtained from multiple replicates of the real-time PCR.
Figure Legend Snippet: Analysis of TaeIF5A1 expression in response to different abiotic stresses. A–E. Two-month-old seedlings were watered with one of the following solutions containing 0.4 M NaCl, 20% PEG6000, 0.3 M NaHCO 3 , 150 μM CdCl 2 , or 150 μM ABA for the indicated times. The relative expression level = log2 (transcription level under stress treatment / transcription level under control conditions). The error bars were obtained from multiple replicates of the real-time PCR.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Expression patterns of TaeIF5A1 and its upstream regulator genes (TaRAV and TaWRKY) and their Arabidopsis homologs. A–B. Expression patterns of TaeIF5A1 and its upstream regulator genes ( TaRAV and TaWRKY ) in the roots, stems and leaves of 2-month-old T . hispida seedlings subjected to 150 μM ABA or 20% PEG6000 treatment. C–D. Expression patterns of the AT1G13950, AT1G68840 and AT1G13960 genes in the roots and leaves of three week-old Arabidopsis seedlings subjected to 100 μM ABA or 20% PEG6000 treatment. AT1G13950, AT1G68840 and AT1G13960 in Arabidopsis are homolog to TaeIF5A1 , TaRAV and TaWRKY , respectively. The relative expression level = log2 (transcription level under stress treatment / transcription level under control conditions). The error bars were obtained from multiple replicates of the real-time PCR.
Figure Legend Snippet: Expression patterns of TaeIF5A1 and its upstream regulator genes (TaRAV and TaWRKY) and their Arabidopsis homologs. A–B. Expression patterns of TaeIF5A1 and its upstream regulator genes ( TaRAV and TaWRKY ) in the roots, stems and leaves of 2-month-old T . hispida seedlings subjected to 150 μM ABA or 20% PEG6000 treatment. C–D. Expression patterns of the AT1G13950, AT1G68840 and AT1G13960 genes in the roots and leaves of three week-old Arabidopsis seedlings subjected to 100 μM ABA or 20% PEG6000 treatment. AT1G13950, AT1G68840 and AT1G13960 in Arabidopsis are homolog to TaeIF5A1 , TaRAV and TaWRKY , respectively. The relative expression level = log2 (transcription level under stress treatment / transcription level under control conditions). The error bars were obtained from multiple replicates of the real-time PCR.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

22) Product Images from "Embryonic frog epidermis: a model for the study of cell-cell interactions in the development of mucociliary disease"

Article Title: Embryonic frog epidermis: a model for the study of cell-cell interactions in the development of mucociliary disease

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.006494

Impact of foxi1e knockdown on the mucociliary epidermis. (A) Diagrammatic representation of foxi1e , showing the position of the ATG MO (red) and sp MO (blue). E1, exon 1; E2, exon 2; I, intron. (B) Reverse transcriptase (RT)-PCR with ornithine decarboxylase ( odc ) control and foxi1e primers, showing that abnormal splicing of foxi1e is observed when a foxi1e sp MO is injected. Asterisk marks reduction in foxi1e splice product in foxi1e splice morphants compared with control. (C) Injection of foxi1e sp MO reduces (11/11) and injection of foxi1e ATG MO abolishes (11/11) the expression of v1a , whereas MOC has no effect (0/9) at early tadpole stage as shown by whole-mount in situ hybridisation. Neither foxi1e sp MO (0/13), foxi1e ATG MO (0/11) or MOC (0/10) had an effect on expression of the ciliated cell marker α-1-tubulin. Scale bars: 500 μm. (D) Embryos were injected with MOC or foxi1e ATG MO (20 ng each) and analysed as indicated. In situ hybridisation with foxi1e probe shows that the spotted expression of foxi1e is reduced (9/9) in foxi1e morphants, whereas MOC has no effect (0/11). Scale bars: 500 μm. Immunostaining of tadpole epidermis with anti-foxi1e antibody shows that the protein expression is reduced in foxi1e morphants (11/12) but not in the MOC controls (0/10). In situ hybridisation with α -1-tubulin probe (black), combined with anti-xeel antibody staining (red), as indicated, reveals that, in the foxi1e morphants, the number of cells that are not positive for either α-1-tubulin or xeel is greatly reduced compared with controls. This suggests that the missing cells are ionocytes. Actin staining (phalloidin–Alexa-Fluor-488; green) combined with anti-acetylated α-tubulin antibody (red) reveals that epidermal cells are arranged in a rosette pattern around each ciliated cell in the foxi1e -ATG-MO-injected epidermis, but in the MOC control (stained with anti-ZO-1 and acetylated α-tubulin), this rosette formation is broken by the insertion of ionocytes (inset shows higher-magnification view). See text for details. Scale bars: 50 μm. (E) Immunostaining for v1a (green), ca12 (green) and acetylated α-tubulin (red) in the combinations indicated confirms that ionocyte markers are missing in foxi1e -ATG-MO-injected embryos (34/35; n =5 experiments) but not in MOC controls (0/35; n =5 experiments). Scale bars: 50 μm. High-magnification images (zoom; scale bar: 20 μm) reveal that ciliated cells are abnormal in the foxi1e morphants. Bar graph shows quantification of the experiments looking at defective ciliated cells ( n =4 experiments). Defective ciliated cells were evident in the majority of foxi1e ATG morphant embryos (42/47), but not in MOC-treated embryos (2/32). ** P =0.0018.
Figure Legend Snippet: Impact of foxi1e knockdown on the mucociliary epidermis. (A) Diagrammatic representation of foxi1e , showing the position of the ATG MO (red) and sp MO (blue). E1, exon 1; E2, exon 2; I, intron. (B) Reverse transcriptase (RT)-PCR with ornithine decarboxylase ( odc ) control and foxi1e primers, showing that abnormal splicing of foxi1e is observed when a foxi1e sp MO is injected. Asterisk marks reduction in foxi1e splice product in foxi1e splice morphants compared with control. (C) Injection of foxi1e sp MO reduces (11/11) and injection of foxi1e ATG MO abolishes (11/11) the expression of v1a , whereas MOC has no effect (0/9) at early tadpole stage as shown by whole-mount in situ hybridisation. Neither foxi1e sp MO (0/13), foxi1e ATG MO (0/11) or MOC (0/10) had an effect on expression of the ciliated cell marker α-1-tubulin. Scale bars: 500 μm. (D) Embryos were injected with MOC or foxi1e ATG MO (20 ng each) and analysed as indicated. In situ hybridisation with foxi1e probe shows that the spotted expression of foxi1e is reduced (9/9) in foxi1e morphants, whereas MOC has no effect (0/11). Scale bars: 500 μm. Immunostaining of tadpole epidermis with anti-foxi1e antibody shows that the protein expression is reduced in foxi1e morphants (11/12) but not in the MOC controls (0/10). In situ hybridisation with α -1-tubulin probe (black), combined with anti-xeel antibody staining (red), as indicated, reveals that, in the foxi1e morphants, the number of cells that are not positive for either α-1-tubulin or xeel is greatly reduced compared with controls. This suggests that the missing cells are ionocytes. Actin staining (phalloidin–Alexa-Fluor-488; green) combined with anti-acetylated α-tubulin antibody (red) reveals that epidermal cells are arranged in a rosette pattern around each ciliated cell in the foxi1e -ATG-MO-injected epidermis, but in the MOC control (stained with anti-ZO-1 and acetylated α-tubulin), this rosette formation is broken by the insertion of ionocytes (inset shows higher-magnification view). See text for details. Scale bars: 50 μm. (E) Immunostaining for v1a (green), ca12 (green) and acetylated α-tubulin (red) in the combinations indicated confirms that ionocyte markers are missing in foxi1e -ATG-MO-injected embryos (34/35; n =5 experiments) but not in MOC controls (0/35; n =5 experiments). Scale bars: 50 μm. High-magnification images (zoom; scale bar: 20 μm) reveal that ciliated cells are abnormal in the foxi1e morphants. Bar graph shows quantification of the experiments looking at defective ciliated cells ( n =4 experiments). Defective ciliated cells were evident in the majority of foxi1e ATG morphant embryos (42/47), but not in MOC-treated embryos (2/32). ** P =0.0018.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Injection, Expressing, In Situ, Hybridization, Marker, Immunostaining, Staining

23) Product Images from "A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions"

Article Title: A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions

Journal: Science Advances

doi: 10.1126/sciadv.aay9634

Characterization of FgGMTV1 infecting F. graminearum strain HB58 and composition of the isolated virus particles. ( A ) Colony morphology of virus-carrying field strain HB58 of F. graminearum after 3 days of culture on potato dextrose agar (PDA) in the dark. ( B ) Viral particles observed under transmission electron microscopy. Scale bar, 50 nm. ( C ) One percent agarose gel electrophoresis of DNA extracted from virus particles. bp, base pair. ( D ) Electrophoresis analysis of enzyme-treated nucleic acid samples on 1% agarose gel. The samples were treated with DNase I, exonuclease III, S1 nuclease, and exonuclease I, respectively. ( E ) SDS–polyacrylamide gel electrophoresis analysis of purified virus particles from strain HB58 showing three protein bands. ( F ) Southern blot analyses of nucleic acids extracted from viral particles. Genomic DNA extracted from strain HB58 was used as a positive control. The forms of the viral DNA are indicated as OC dsDNA (open circular dsDNA), SC dsDNA (supercoiled dsDNA), and circular ssDNA. Three DNA fragments (probe A, probe B, and probe C, specific for DNA-A, DNA-B, and DNA-C, respectively) were PCR-amplified, labeled with DIG, and used as DNA probes. ( G ) Genome organization of the three components of FgGMTV1. The single ORF of each DNA component is displayed as a thick arrow. The positions of the potential stem-loop structure, common region major (CR-M) and common region stem-loop (CR-SL) are indicated. Photo credit: Pengfei Li, Institute of Plant Protection, Chinese Academy of Agricultural Sciences.
Figure Legend Snippet: Characterization of FgGMTV1 infecting F. graminearum strain HB58 and composition of the isolated virus particles. ( A ) Colony morphology of virus-carrying field strain HB58 of F. graminearum after 3 days of culture on potato dextrose agar (PDA) in the dark. ( B ) Viral particles observed under transmission electron microscopy. Scale bar, 50 nm. ( C ) One percent agarose gel electrophoresis of DNA extracted from virus particles. bp, base pair. ( D ) Electrophoresis analysis of enzyme-treated nucleic acid samples on 1% agarose gel. The samples were treated with DNase I, exonuclease III, S1 nuclease, and exonuclease I, respectively. ( E ) SDS–polyacrylamide gel electrophoresis analysis of purified virus particles from strain HB58 showing three protein bands. ( F ) Southern blot analyses of nucleic acids extracted from viral particles. Genomic DNA extracted from strain HB58 was used as a positive control. The forms of the viral DNA are indicated as OC dsDNA (open circular dsDNA), SC dsDNA (supercoiled dsDNA), and circular ssDNA. Three DNA fragments (probe A, probe B, and probe C, specific for DNA-A, DNA-B, and DNA-C, respectively) were PCR-amplified, labeled with DIG, and used as DNA probes. ( G ) Genome organization of the three components of FgGMTV1. The single ORF of each DNA component is displayed as a thick arrow. The positions of the potential stem-loop structure, common region major (CR-M) and common region stem-loop (CR-SL) are indicated. Photo credit: Pengfei Li, Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

Techniques Used: Isolation, Transmission Assay, Electron Microscopy, Agarose Gel Electrophoresis, Electrophoresis, Polyacrylamide Gel Electrophoresis, Purification, Southern Blot, Positive Control, Polymerase Chain Reaction, Amplification, Labeling

24) Product Images from "A Novel Soybean ERF Transcription Factor, GmERF113, Increases Resistance to Phytophthora sojae Infection in Soybean"

Article Title: A Novel Soybean ERF Transcription Factor, GmERF113, Increases Resistance to Phytophthora sojae Infection in Soybean

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.00299

Responses of living of GmERF113 transgenic soybean plants to P. sojae . (A) Quantitative real-time PCR (qRT-PCR) analysis of GmERF113 expression levels in T 3 transgenic soybean plants. (B) Disease symptoms on the leaves of T 3 transgenic and non-transgenic lines infected with P. sojae race 1 inoculum at 2 and 4 days. (C) The lesion areas of the transgenic and non-transgenic lines were determined 4 days after inoculation with P. sojae . (D) qRT-PCR analysis of GmERF113 expression levels in T 4 transgenic soybean plants. (E) Disease symptoms on the cotyledons of T 4 transgenic and non-transgenic lines 48 h after treatment with P. sojae zoospore suspension. (F) qRT-PCR analysis of P. sojae relative biomass based on the transcript level of the P. sojae TEF1 gene in infected cotyledons 48 h after incubation with P. sojae zoospore suspension. The experiment was performed using three biological replicates with three technical replicates each and statistically analyzed using Student’s t -tests ( ∗∗ P
Figure Legend Snippet: Responses of living of GmERF113 transgenic soybean plants to P. sojae . (A) Quantitative real-time PCR (qRT-PCR) analysis of GmERF113 expression levels in T 3 transgenic soybean plants. (B) Disease symptoms on the leaves of T 3 transgenic and non-transgenic lines infected with P. sojae race 1 inoculum at 2 and 4 days. (C) The lesion areas of the transgenic and non-transgenic lines were determined 4 days after inoculation with P. sojae . (D) qRT-PCR analysis of GmERF113 expression levels in T 4 transgenic soybean plants. (E) Disease symptoms on the cotyledons of T 4 transgenic and non-transgenic lines 48 h after treatment with P. sojae zoospore suspension. (F) qRT-PCR analysis of P. sojae relative biomass based on the transcript level of the P. sojae TEF1 gene in infected cotyledons 48 h after incubation with P. sojae zoospore suspension. The experiment was performed using three biological replicates with three technical replicates each and statistically analyzed using Student’s t -tests ( ∗∗ P

Techniques Used: Transgenic Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Infection, Incubation

25) Product Images from "Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells"

Article Title: Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0003942

Construction of IgM + memory B cell libraries. a) Donor lymphocytes were isolated by Ficoll-plaque from heparinized blood and stained for the phenotypic markers CD27, CD24 and IgM. CD24 + CD27 + cells were gated and the IgM + cells within this gate sorted directly into Trizol for RNA extraction. b) RT-PCR was performed using a pool of 5′ oligonucleotide primers that cover all VH gene families and a 3′ oligonucleotide primer that anneals in a region of the CH1 domain of Cμ distinct from other immunoglobulin isotypes. c) Using cDNA generated in this way, 10 individual scFv libraries were constructed as described previously [26] . Donors 1020, 1030 and 1050 had been vaccinated with the Dutch 2005 seasonal influenza vaccine 7 days prior to collection of blood. All libraries demonstrated a high percentage of correct scFv ORF's and diversity based on unique HCDR3 sequence.
Figure Legend Snippet: Construction of IgM + memory B cell libraries. a) Donor lymphocytes were isolated by Ficoll-plaque from heparinized blood and stained for the phenotypic markers CD27, CD24 and IgM. CD24 + CD27 + cells were gated and the IgM + cells within this gate sorted directly into Trizol for RNA extraction. b) RT-PCR was performed using a pool of 5′ oligonucleotide primers that cover all VH gene families and a 3′ oligonucleotide primer that anneals in a region of the CH1 domain of Cμ distinct from other immunoglobulin isotypes. c) Using cDNA generated in this way, 10 individual scFv libraries were constructed as described previously [26] . Donors 1020, 1030 and 1050 had been vaccinated with the Dutch 2005 seasonal influenza vaccine 7 days prior to collection of blood. All libraries demonstrated a high percentage of correct scFv ORF's and diversity based on unique HCDR3 sequence.

Techniques Used: Isolation, Staining, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Generated, Construct, Sequencing

PCR screen of individual donor libraries for neutralizing mAbs and donor serology. (a) PCR amplification of cDNA from each donor IgM + memory B cell library using oligonucleotide pairs designed so their 3′ ends specifically anneal in the HCDR1 and HCDR3 regions. Donors are indicated at the top of the figure. The expected size of the amplified fragment is indicated with an arrow. The identity of the bands was confirmed by sequencing (b) Binding and neutralizing activity of donor serum collected at the same time as the B cells used for library construction (note serum was not available for donor 12). IgM and IgG ELISA reactivity was measured against rHA and neutralizing activity against H1N1 (A/Hong Kong/54/98) and H5N1 (A/Vietnam/1203/04). Donor 1020 who was PCR positive for the tested neutralizing mAbs is indicated in bold.
Figure Legend Snippet: PCR screen of individual donor libraries for neutralizing mAbs and donor serology. (a) PCR amplification of cDNA from each donor IgM + memory B cell library using oligonucleotide pairs designed so their 3′ ends specifically anneal in the HCDR1 and HCDR3 regions. Donors are indicated at the top of the figure. The expected size of the amplified fragment is indicated with an arrow. The identity of the bands was confirmed by sequencing (b) Binding and neutralizing activity of donor serum collected at the same time as the B cells used for library construction (note serum was not available for donor 12). IgM and IgG ELISA reactivity was measured against rHA and neutralizing activity against H1N1 (A/Hong Kong/54/98) and H5N1 (A/Vietnam/1203/04). Donor 1020 who was PCR positive for the tested neutralizing mAbs is indicated in bold.

Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

26) Product Images from "HSPA12A is required for adipocyte differentiation and diet-induced obesity through a positive feedback regulation with PPARγ"

Article Title: HSPA12A is required for adipocyte differentiation and diet-induced obesity through a positive feedback regulation with PPARγ

Journal: Cell Death and Differentiation

doi: 10.1038/s41418-019-0300-2

Deficiency of HSPA12A suppressed PPARγ and its target genes linking to adipocyte differentiation in mice. Inguinal WAT were collected from mice that fed with HFD or chow diet for 14 weeks. The expression of the indicated mRNA and proteins was analyzed by Real-time PCR ( a ) and Immunoblotting ( b ), respectively. Data are mean ± SEM, ** P
Figure Legend Snippet: Deficiency of HSPA12A suppressed PPARγ and its target genes linking to adipocyte differentiation in mice. Inguinal WAT were collected from mice that fed with HFD or chow diet for 14 weeks. The expression of the indicated mRNA and proteins was analyzed by Real-time PCR ( a ) and Immunoblotting ( b ), respectively. Data are mean ± SEM, ** P

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

HSPA12A regulated adipocyte differentiation and PPARγ expression in vitro. a , b HSPA12A deficiency suppressed adipocyte differentiation. Differentiation was induced in primary SVF isolated from WT and Hspa12a -/- mice. Lipid droplets were examined by ORO staining ( a ). Expression of mRNA was examined using real-time PCR ( b ). Data are mean ± SEM, ** P
Figure Legend Snippet: HSPA12A regulated adipocyte differentiation and PPARγ expression in vitro. a , b HSPA12A deficiency suppressed adipocyte differentiation. Differentiation was induced in primary SVF isolated from WT and Hspa12a -/- mice. Lipid droplets were examined by ORO staining ( a ). Expression of mRNA was examined using real-time PCR ( b ). Data are mean ± SEM, ** P

Techniques Used: Expressing, In Vitro, Isolation, Mouse Assay, Staining, Real-time Polymerase Chain Reaction

27) Product Images from "Low Intracellular Iron Increases the Stability of Matriptase-2 *"

Article Title: Low Intracellular Iron Increases the Stability of Matriptase-2 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.611913

Iron deficiency does not alter the translation of Tmprss6 mRNA in the rat liver . A, acute and chronic iron deprivation does not alter Tmprss6 mRNA expression in rats. Weanling rats were fed either a control ( Ctrl ) iron diet or an iron-deficient diet ( ID ) for 3 days (day 3; n = 3) or 14 days (day 14; n = 5). Hepcidin ( Hamp ), Tmprss6 ( TM6 ), and mitochondrial aconitase ( m-acon ) mRNA expression in the liver were analyzed by qRT-PCR. Results are expressed as the amount of mRNA relative to β-actin in each sample. B, chronic iron deprivation increases MT2 protein in the liver. MT2 protein levels in the liver tissues from rats with chronic iron deprivation (day 14) as described above in A were detected by Western blot. β-Actin was included as a loading control. C–E, qRT-PCR analysis of m-Acon ( C ), Tmprss6 ( D ), and β-actin ( E ) mRNA in the fractions of polysome fractionation from the liver tissues of rats fed a control ( Ctrl ) iron diet or an iron-deficient diet ( ID ) for 3 days. The amount of mRNA in each fraction was expressed as the percentage of the combined total mRNA in all fractions. Each group consisted of three animals. F–H, qRT-PCR analysis of m-Acon ( F ), Tmprss6 ( G ), and β-actin ( H ) mRNA in the fractions of polysome fractionation from the liver tissues of rats fed a control ( Ctrl ) iron diet or an iron-deficient diet ( ID ) for 14 days. The amount of mRNA in each fraction was expressed as the percentage of the combined total mRNA in all fractions. Each group consisted of five animals. *, p
Figure Legend Snippet: Iron deficiency does not alter the translation of Tmprss6 mRNA in the rat liver . A, acute and chronic iron deprivation does not alter Tmprss6 mRNA expression in rats. Weanling rats were fed either a control ( Ctrl ) iron diet or an iron-deficient diet ( ID ) for 3 days (day 3; n = 3) or 14 days (day 14; n = 5). Hepcidin ( Hamp ), Tmprss6 ( TM6 ), and mitochondrial aconitase ( m-acon ) mRNA expression in the liver were analyzed by qRT-PCR. Results are expressed as the amount of mRNA relative to β-actin in each sample. B, chronic iron deprivation increases MT2 protein in the liver. MT2 protein levels in the liver tissues from rats with chronic iron deprivation (day 14) as described above in A were detected by Western blot. β-Actin was included as a loading control. C–E, qRT-PCR analysis of m-Acon ( C ), Tmprss6 ( D ), and β-actin ( E ) mRNA in the fractions of polysome fractionation from the liver tissues of rats fed a control ( Ctrl ) iron diet or an iron-deficient diet ( ID ) for 3 days. The amount of mRNA in each fraction was expressed as the percentage of the combined total mRNA in all fractions. Each group consisted of three animals. F–H, qRT-PCR analysis of m-Acon ( F ), Tmprss6 ( G ), and β-actin ( H ) mRNA in the fractions of polysome fractionation from the liver tissues of rats fed a control ( Ctrl ) iron diet or an iron-deficient diet ( ID ) for 14 days. The amount of mRNA in each fraction was expressed as the percentage of the combined total mRNA in all fractions. Each group consisted of five animals. *, p

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

Lack of change in Tmprss6 mRNA by BMP6, ID1, the BMP signaling, or iron. A, BMP6 did not induce TMPRSS6 mRNA expression in HepG2 cells. HepG2 cells were incubated in the presence of BMP6 at 0, 5, 25, and 50 ng/ml for 18 h, followed by qRT-PCR analysis of hepcidin, TMPRSS6, ID1 , and SMAD7 mRNA. The results are expressed as the amount of mRNA relative to β-actin in each sample. Results from four individual experiments are presented. B, increases in Bmp6 and Id1 expression did not induce Tmprss6 expression in mice. Eight-week-old Hjv- null male mice were injected with AAV8-Hjv vector containing a strong liver-specific promoter (−/− Hjv ) or the carrier vehicle (−/−). After 2 weeks, mice were euthanized for qRT-PCR analysis of Bmp6, Id1, Smad7, Tmprss6, and hepcidin mRNA in the liver. The results are expressed as the amount of mRNA relative to β-actin in each sample. Strain-, age-, and gender-matched wild-type ( wt ) mice were included as controls. Each group consisted of five animals. C and D, chronic iron load did not alter Tmprss6 expression in hepatocytes and the liver. Wild-type (9 weeks old) 129/S male mice were fed either a high iron rodent diet with 2% carbonyl iron (TD.08496; Harlan Laboratories) or a control iron rodent diet with 48 ppm iron (TD.80394; Harlan Laboratories) for 3 weeks. Tmprss6 mRNA in the whole liver and the isolated hepatocytes ( HC ), KC, SEC, and HSC ( C ), as well as hepcidin, Id1 , and Smad7 mRNA from the isolated hepatocytes of these mice ( D ) was analyzed by qRT-PCR. The results are expressed as the amount of mRNA relative to β-actin in each sample. Each group consisted of five animals. **, p
Figure Legend Snippet: Lack of change in Tmprss6 mRNA by BMP6, ID1, the BMP signaling, or iron. A, BMP6 did not induce TMPRSS6 mRNA expression in HepG2 cells. HepG2 cells were incubated in the presence of BMP6 at 0, 5, 25, and 50 ng/ml for 18 h, followed by qRT-PCR analysis of hepcidin, TMPRSS6, ID1 , and SMAD7 mRNA. The results are expressed as the amount of mRNA relative to β-actin in each sample. Results from four individual experiments are presented. B, increases in Bmp6 and Id1 expression did not induce Tmprss6 expression in mice. Eight-week-old Hjv- null male mice were injected with AAV8-Hjv vector containing a strong liver-specific promoter (−/− Hjv ) or the carrier vehicle (−/−). After 2 weeks, mice were euthanized for qRT-PCR analysis of Bmp6, Id1, Smad7, Tmprss6, and hepcidin mRNA in the liver. The results are expressed as the amount of mRNA relative to β-actin in each sample. Strain-, age-, and gender-matched wild-type ( wt ) mice were included as controls. Each group consisted of five animals. C and D, chronic iron load did not alter Tmprss6 expression in hepatocytes and the liver. Wild-type (9 weeks old) 129/S male mice were fed either a high iron rodent diet with 2% carbonyl iron (TD.08496; Harlan Laboratories) or a control iron rodent diet with 48 ppm iron (TD.80394; Harlan Laboratories) for 3 weeks. Tmprss6 mRNA in the whole liver and the isolated hepatocytes ( HC ), KC, SEC, and HSC ( C ), as well as hepcidin, Id1 , and Smad7 mRNA from the isolated hepatocytes of these mice ( D ) was analyzed by qRT-PCR. The results are expressed as the amount of mRNA relative to β-actin in each sample. Each group consisted of five animals. **, p

Techniques Used: Expressing, Incubation, Quantitative RT-PCR, Mouse Assay, Injection, Plasmid Preparation, Isolation, Size-exclusion Chromatography

28) Product Images from "Decreased expression of 17?-hydroxysteroid dehydrogenase type 1 is associated with DNA hypermethylation in colorectal cancer located in the proximal colon"

Article Title: Decreased expression of 17?-hydroxysteroid dehydrogenase type 1 is associated with DNA hypermethylation in colorectal cancer located in the proximal colon

Journal: BMC Cancer

doi: 10.1186/1471-2407-11-522

Bisulfite sequencing of the CpG rich region fragment (A) and binding of Pol II to HSD17B1 promoter (B) in HT29 and SW707 colorectal cancer cells treated with 5-dAzaC . HT29 and SW707 cells were incubated for 48 h either in the absence or in the presence of 5-dAzaC (1.00 μM). The cells were then used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. The CpG rich region containing 31 CpG dinucleotides (chr17: 37 953 392-37 953 917) was then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence (Additional file 1 , Additional file 2 ). The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from ten positive bacterial clones was used for commercial sequencing. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and BDPC web server, respectively [ 22 , 23 ]. Black and grey boxes represent methylated and unmethylated CpG dinucleotide, respectively. White boxes correspond to undetermined CpG dinucleotide. For the ChIP assay, HT29 and SW707 cells were incubated for 0, 6, 12, 24, 36, and 48 h either in the absence or in the presence of 5-dAzaC (1.00 μM). After incubation, cells were used for ChIP analysis with anti-Pol II Ab. RQ-PCR was carried out by pairs of primers complementary to the HSD17B1 promoter for the HT29 (-◆-) and SW707 (...▲...) cell lines (Additional file 1 , Additional file 2 ). Data are expressed as a percentage of the HSD17B1 promoter occupied by Pol II. The results are presented as the mean ± SE from three independent experiments.
Figure Legend Snippet: Bisulfite sequencing of the CpG rich region fragment (A) and binding of Pol II to HSD17B1 promoter (B) in HT29 and SW707 colorectal cancer cells treated with 5-dAzaC . HT29 and SW707 cells were incubated for 48 h either in the absence or in the presence of 5-dAzaC (1.00 μM). The cells were then used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. The CpG rich region containing 31 CpG dinucleotides (chr17: 37 953 392-37 953 917) was then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence (Additional file 1 , Additional file 2 ). The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from ten positive bacterial clones was used for commercial sequencing. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and BDPC web server, respectively [ 22 , 23 ]. Black and grey boxes represent methylated and unmethylated CpG dinucleotide, respectively. White boxes correspond to undetermined CpG dinucleotide. For the ChIP assay, HT29 and SW707 cells were incubated for 0, 6, 12, 24, 36, and 48 h either in the absence or in the presence of 5-dAzaC (1.00 μM). After incubation, cells were used for ChIP analysis with anti-Pol II Ab. RQ-PCR was carried out by pairs of primers complementary to the HSD17B1 promoter for the HT29 (-◆-) and SW707 (...▲...) cell lines (Additional file 1 , Additional file 2 ). Data are expressed as a percentage of the HSD17B1 promoter occupied by Pol II. The results are presented as the mean ± SE from three independent experiments.

Techniques Used: Methylation Sequencing, Binding Assay, Incubation, DNA Extraction, Amplification, Modification, Sequencing, Polymerase Chain Reaction, Purification, Clone Assay, Plasmid Preparation, Isolation, Software, Methylation, Chromatin Immunoprecipitation

Bisulfite sequencing of DNA CpG rich region in primary tissue samples from patients with CRC in the proximal (A) and distal (B) colon . The primary cancerous and histopathologically unchanged tissues from the same patients with cancer in the proximal (P1-P5) and distal colon (P6-P10) were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. The HSD17B1 region containing 31 CpG dinucleotides (chr17: 37 953 392-37 953 917) was then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence (Additional file 1 , Additional file 2 ). The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from ten positive bacterial clones was used for commercial sequencing. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and BDPC web server, respectively [ 22 , 23 ]. Black and grey boxes represent methylated and unmethylated CpG dinucleotide, respectively. White boxes correspond to undetermined CpG dinucleotide. The legend with grey scale corresponds to average methylation in (P1-P5) and (P6-P10).
Figure Legend Snippet: Bisulfite sequencing of DNA CpG rich region in primary tissue samples from patients with CRC in the proximal (A) and distal (B) colon . The primary cancerous and histopathologically unchanged tissues from the same patients with cancer in the proximal (P1-P5) and distal colon (P6-P10) were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. The HSD17B1 region containing 31 CpG dinucleotides (chr17: 37 953 392-37 953 917) was then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence (Additional file 1 , Additional file 2 ). The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from ten positive bacterial clones was used for commercial sequencing. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and BDPC web server, respectively [ 22 , 23 ]. Black and grey boxes represent methylated and unmethylated CpG dinucleotide, respectively. White boxes correspond to undetermined CpG dinucleotide. The legend with grey scale corresponds to average methylation in (P1-P5) and (P6-P10).

Techniques Used: Methylation Sequencing, DNA Extraction, Amplification, Modification, Sequencing, Polymerase Chain Reaction, Purification, Clone Assay, Plasmid Preparation, Isolation, Software, Methylation

29) Product Images from "Chemoresistance to Valproate Treatment of Bovine Leukemia Virus-Infected Sheep; Identification of Improved HDAC Inhibitors"

Article Title: Chemoresistance to Valproate Treatment of Bovine Leukemia Virus-Infected Sheep; Identification of Improved HDAC Inhibitors

Journal: Pathogens

doi: 10.3390/pathogens1020065

BLV proviral integrity and integration sites during VPA treatment and relapse: A. DNA was extracted from BLV-infected sheep PBMCs (2213, 3002, 3003, 4213, 4535) isolated just before VPA treatment at day 0 (B) and at the end of the relapse period (R). The full length proviral sequence was amplified by PCR using primers located in the 5' and 3' LTRs. PCR amplification products were migrated onto an agarose gel. The molecular weight of the amplicon is indicated in kilobases (8 Kb). B. Southern blot hybridization using a BLV probe of genomic DNA digested with EcoRI. The molecular weight markers (in Kb) are indicated on the left. Lanes correspond to those of panel A.
Figure Legend Snippet: BLV proviral integrity and integration sites during VPA treatment and relapse: A. DNA was extracted from BLV-infected sheep PBMCs (2213, 3002, 3003, 4213, 4535) isolated just before VPA treatment at day 0 (B) and at the end of the relapse period (R). The full length proviral sequence was amplified by PCR using primers located in the 5' and 3' LTRs. PCR amplification products were migrated onto an agarose gel. The molecular weight of the amplicon is indicated in kilobases (8 Kb). B. Southern blot hybridization using a BLV probe of genomic DNA digested with EcoRI. The molecular weight markers (in Kb) are indicated on the left. Lanes correspond to those of panel A.

Techniques Used: Infection, Isolation, Sequencing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Southern Blot, Hybridization

BLV proviral loads during VPA treatment and relapse: The proviral loads (empty squares), represented as numbers of viral copies per 10 3 B-lymphocytes, were measured by real time PCR using genomic DNA isolated from sheep peripheral blood mononuclear cells (PBMCs). Data result from triplicate measurements (error bars represent means ± standard deviations). Absolute leukocyte numbers are indicated as reference (dotted line).
Figure Legend Snippet: BLV proviral loads during VPA treatment and relapse: The proviral loads (empty squares), represented as numbers of viral copies per 10 3 B-lymphocytes, were measured by real time PCR using genomic DNA isolated from sheep peripheral blood mononuclear cells (PBMCs). Data result from triplicate measurements (error bars represent means ± standard deviations). Absolute leukocyte numbers are indicated as reference (dotted line).

Techniques Used: Real-time Polymerase Chain Reaction, Isolation

30) Product Images from "Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants"

Article Title: Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2016.01572

Verification of designed TALENs in N. benthamiana infiltration. (Top panel) Target vector pSIM2167 of TALENs for N. benthamiana infiltration. A 60 bp DNA sequence from StUbi7 5′ intron was inserted between the first and second amino acid codons of the GUS gene. There is an in frame stop codon (dark gray highlighted) in the spacer region between the two recognition sites (light gray highlighted) which will abolish the GUS protein translation. TALEN-mediated DNA cleavage and subsequent NHEJ based repairing will destroy the stop codon and translation of the GUS will be in frame and restore GUS activity. An AluI restriction enzyme site (underlined) was also included in the spacer to facilitate detection of TALEN-mediated indels. (Middle and bottom panels): The functionality of designed TALEN in transient assay. Target construct was Agro -infiltrated into N. benthamiana leaves alone or together with the TALEN construct. Forty-eight hours after infiltration, leaf tissue at the infiltrated site was collected and stained with GUS staining solution and de-stained with ethanol for microscopic examination. DNA was isolated from co-infiltrated tissue, digested with AluI , PCR amplified, cloned and sequenced. (Middle left panel): Target construct pSIM2167 alone. (Middle right panel): Target construct pSIM2167 co-infiltrated with TALEN construct pSIM2170. (Bottom panel): Sequences of target region with various modifications. The number in the parenthesis represents how many times the modification occurred in the 50 clones sequenced. The numbers on the left are identifiers of the respective clones.
Figure Legend Snippet: Verification of designed TALENs in N. benthamiana infiltration. (Top panel) Target vector pSIM2167 of TALENs for N. benthamiana infiltration. A 60 bp DNA sequence from StUbi7 5′ intron was inserted between the first and second amino acid codons of the GUS gene. There is an in frame stop codon (dark gray highlighted) in the spacer region between the two recognition sites (light gray highlighted) which will abolish the GUS protein translation. TALEN-mediated DNA cleavage and subsequent NHEJ based repairing will destroy the stop codon and translation of the GUS will be in frame and restore GUS activity. An AluI restriction enzyme site (underlined) was also included in the spacer to facilitate detection of TALEN-mediated indels. (Middle and bottom panels): The functionality of designed TALEN in transient assay. Target construct was Agro -infiltrated into N. benthamiana leaves alone or together with the TALEN construct. Forty-eight hours after infiltration, leaf tissue at the infiltrated site was collected and stained with GUS staining solution and de-stained with ethanol for microscopic examination. DNA was isolated from co-infiltrated tissue, digested with AluI , PCR amplified, cloned and sequenced. (Middle left panel): Target construct pSIM2167 alone. (Middle right panel): Target construct pSIM2167 co-infiltrated with TALEN construct pSIM2170. (Bottom panel): Sequences of target region with various modifications. The number in the parenthesis represents how many times the modification occurred in the 50 clones sequenced. The numbers on the left are identifiers of the respective clones.

Techniques Used: TALENs, Plasmid Preparation, Sequencing, Non-Homologous End Joining, Activity Assay, Construct, Staining, Isolation, Polymerase Chain Reaction, Amplification, Clone Assay, Modification

Identification of targeted integration events. DNA from herbicide resistant transgenic lines were isolated for PCR and Southern analyses. (Top panel) Schematic of the Ubi7 locus after site-specific gene integration. Primers 1: within the endogenous Ubi7 promoter and 2: within the mStALS gene coding region are indicated by arrows. Two HindIII restriction enzyme sites, one in the endogenous Ubi7 promoter and one in the mStALS coding region are also labeled. (Middle panel) : PCR using primers 1 and 2 detects site-specific integration events. (Bottom panel) : Southern blot using StALS probe detects the site-specific integration band at predicted size (boxed). The size of dig-labeled ladders were marked as kb (M: Dig-labeled marker 7, M2: Dig-labeled marker 2).
Figure Legend Snippet: Identification of targeted integration events. DNA from herbicide resistant transgenic lines were isolated for PCR and Southern analyses. (Top panel) Schematic of the Ubi7 locus after site-specific gene integration. Primers 1: within the endogenous Ubi7 promoter and 2: within the mStALS gene coding region are indicated by arrows. Two HindIII restriction enzyme sites, one in the endogenous Ubi7 promoter and one in the mStALS coding region are also labeled. (Middle panel) : PCR using primers 1 and 2 detects site-specific integration events. (Bottom panel) : Southern blot using StALS probe detects the site-specific integration band at predicted size (boxed). The size of dig-labeled ladders were marked as kb (M: Dig-labeled marker 7, M2: Dig-labeled marker 2).

Techniques Used: Transgenic Assay, Isolation, Polymerase Chain Reaction, Labeling, Southern Blot, Marker

Schematic of strategy for the targeted T-DNA insertion. The TALEN vector contains a pair of TALENs targeting the endogenous potato Ubi7 intron immediately 5′ to the initiation codon for the first ubiquitin monomer. The LB was removed and an ipt overexpression cassette was put next to the TALEN expression cassettes on the left side of T-DNA. The donor vector contains a promoter-less mutated potato ALS gene ( mStALS ) next to the RB and the nptII cassette. Upon Agrobacterium -mediated transformation, expression of TALENs generate DSB at the endogenous target, once the donor T-DNA is inserted into this endogenous site, the endogenous promoter will drive the expression of mStALS expression and resulted targeted events can be selected out on herbicide containing medium. The targeted events can also be PCR confirmed by using a primer in the mStALS gene (opened arrow) paired with a primer within the endogenous Ubi7 promoter (solid arrow).
Figure Legend Snippet: Schematic of strategy for the targeted T-DNA insertion. The TALEN vector contains a pair of TALENs targeting the endogenous potato Ubi7 intron immediately 5′ to the initiation codon for the first ubiquitin monomer. The LB was removed and an ipt overexpression cassette was put next to the TALEN expression cassettes on the left side of T-DNA. The donor vector contains a promoter-less mutated potato ALS gene ( mStALS ) next to the RB and the nptII cassette. Upon Agrobacterium -mediated transformation, expression of TALENs generate DSB at the endogenous target, once the donor T-DNA is inserted into this endogenous site, the endogenous promoter will drive the expression of mStALS expression and resulted targeted events can be selected out on herbicide containing medium. The targeted events can also be PCR confirmed by using a primer in the mStALS gene (opened arrow) paired with a primer within the endogenous Ubi7 promoter (solid arrow).

Techniques Used: Plasmid Preparation, TALENs, Over Expression, Expressing, Transformation Assay, Polymerase Chain Reaction

31) Product Images from "Fatty Acid-and Retinol-Binding Protein, Mj-FAR-1 Induces Tomato Host Susceptibility to Root-Knot Nematodes"

Article Title: Fatty Acid-and Retinol-Binding Protein, Mj-FAR-1 Induces Tomato Host Susceptibility to Root-Knot Nematodes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064586

Constitutive expression of mj-far -1 in tomato hairy roots increases roots susceptibility to infection by the RKN M. javanica . A. RT-PCR confirmation of mj-far-1 (upper gel) and kanR (lower gel) expression in tomato hairy roots lines far -1 E1.1, far -1 E 7.1, far-1 RNAi3.2 and far-1 RNAi11.5 compared with the respective control Vector 1.1 and WT 870. The expected size of the PCR product is 96 bp for the mj-far -1 and 81 for kanR . RT-PCR was performed on total RNA isolated from non infected transformed tomato hairy roots and WT 870 roots. B. Increased susceptibility of tomato hairy roots expressing mj-far -1 ( far-1 E1.1 and far-1 E7.1) is accompanied by expanded galling production compared with mj-far-1 dsRNA-expressing tomato hairy root lines ( far-1 RNAi3.2 and far-1 RNAi11.5) and vector control (Vector 1.1. and Vector 11.5). C. Meloidogyne susceptibility/resistance of vector transformed roots and transgenic tomato roots expressing mj-far-1 , or mj-far-1 dsRNA-expressing lines, was measured as nematode developmental stages counted at 15 and 28 DAI. Roots were inoculated with 200 sterile pre-parasitic J2s and then assessed for juveniles, young females and mature females under the dissecting scope following staining with acid fuchsin dye. Note the significant ( P ≤0.05) increase in percentage of young females at 15DAI and increase in percentage of mature female at 28DAI in roots overexpressing mj-far-1 in comparison with vector control roots. Data are expressed as means of 25 plants from each line; the experiment was repeated three times, giving consistent results. The percentage of each developmental stage is represented by a mean ± standard error. Different letters above the bars denote a significant difference ( P ≤0.05, analysis of variance) between tomato roots lines analyzed by Tukey-Kramer multiple comparison tests.
Figure Legend Snippet: Constitutive expression of mj-far -1 in tomato hairy roots increases roots susceptibility to infection by the RKN M. javanica . A. RT-PCR confirmation of mj-far-1 (upper gel) and kanR (lower gel) expression in tomato hairy roots lines far -1 E1.1, far -1 E 7.1, far-1 RNAi3.2 and far-1 RNAi11.5 compared with the respective control Vector 1.1 and WT 870. The expected size of the PCR product is 96 bp for the mj-far -1 and 81 for kanR . RT-PCR was performed on total RNA isolated from non infected transformed tomato hairy roots and WT 870 roots. B. Increased susceptibility of tomato hairy roots expressing mj-far -1 ( far-1 E1.1 and far-1 E7.1) is accompanied by expanded galling production compared with mj-far-1 dsRNA-expressing tomato hairy root lines ( far-1 RNAi3.2 and far-1 RNAi11.5) and vector control (Vector 1.1. and Vector 11.5). C. Meloidogyne susceptibility/resistance of vector transformed roots and transgenic tomato roots expressing mj-far-1 , or mj-far-1 dsRNA-expressing lines, was measured as nematode developmental stages counted at 15 and 28 DAI. Roots were inoculated with 200 sterile pre-parasitic J2s and then assessed for juveniles, young females and mature females under the dissecting scope following staining with acid fuchsin dye. Note the significant ( P ≤0.05) increase in percentage of young females at 15DAI and increase in percentage of mature female at 28DAI in roots overexpressing mj-far-1 in comparison with vector control roots. Data are expressed as means of 25 plants from each line; the experiment was repeated three times, giving consistent results. The percentage of each developmental stage is represented by a mean ± standard error. Different letters above the bars denote a significant difference ( P ≤0.05, analysis of variance) between tomato roots lines analyzed by Tukey-Kramer multiple comparison tests.

Techniques Used: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Polymerase Chain Reaction, Isolation, Transformation Assay, Transgenic Assay, Staining

32) Product Images from "YB-1, the E2F Pathway, and Regulation of Tumor Cell Growth"

Article Title: YB-1, the E2F Pathway, and Regulation of Tumor Cell Growth

Journal: JNCI Journal of the National Cancer Institute

doi: 10.1093/jnci/djr512

Chromatin immunoprecipitation of E2F1 target gene promoters using YB-1 antibody. Chromatin from MCF-7 cells was cross-linked to fix bound proteins to the DNA. Cells were lysed and the chromatin was incubated with a YB-1 antibody to immunoprecipitate promoters bound by YB-1. Polymerase chain reaction was then performed to amplify promoter fragments of known E2F1-regulated genes to determine whether they were bound by YB-1. As controls, MCF-7 cells were transfected with siYB-1 72 hours before they were harvested to deplete YB-1, or chromatin was precleared with an E2F1 antibody to remove E2F1-bound promoters before incubation with YB-1 antibody. Input = MCF-7 DNA before immunoprecipitation; Beads = protein G beads in the absence of DNA; IgG = chromatin immunoprecipitation with the IgG negative control antibody; YB-1 ChIP = promoters immunoprecipitated with the YB-1 antibody; YB-1 = YB-1 antibody only; siYB-1 = cells pretreated with siYB-1; E2F1 = cells preincubated with E2F1 antibody. Figure shows typical results obtained from at least three independent experiments.
Figure Legend Snippet: Chromatin immunoprecipitation of E2F1 target gene promoters using YB-1 antibody. Chromatin from MCF-7 cells was cross-linked to fix bound proteins to the DNA. Cells were lysed and the chromatin was incubated with a YB-1 antibody to immunoprecipitate promoters bound by YB-1. Polymerase chain reaction was then performed to amplify promoter fragments of known E2F1-regulated genes to determine whether they were bound by YB-1. As controls, MCF-7 cells were transfected with siYB-1 72 hours before they were harvested to deplete YB-1, or chromatin was precleared with an E2F1 antibody to remove E2F1-bound promoters before incubation with YB-1 antibody. Input = MCF-7 DNA before immunoprecipitation; Beads = protein G beads in the absence of DNA; IgG = chromatin immunoprecipitation with the IgG negative control antibody; YB-1 ChIP = promoters immunoprecipitated with the YB-1 antibody; YB-1 = YB-1 antibody only; siYB-1 = cells pretreated with siYB-1; E2F1 = cells preincubated with E2F1 antibody. Figure shows typical results obtained from at least three independent experiments.

Techniques Used: Chromatin Immunoprecipitation, Incubation, Polymerase Chain Reaction, Transfection, Immunoprecipitation, Negative Control

33) Product Images from "Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma"

Article Title: Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma

Journal: International Journal of Otolaryngology

doi: 10.1155/2009/848695

Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.
Figure Legend Snippet: Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.

Techniques Used: Polymerase Chain Reaction, Methylation, DNA Methylation Assay, Positive Control, Marker, Modification, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Electrophoresis

34) Product Images from "Transcription Activator-Like Effector Nuclease (TALEN)-Mediated CLYBL Targeting Enables Enhanced Transgene Expression and One-Step Generation of Dual Reporter Human Induced Pluripotent Stem Cell (iPSC) and Neural Stem Cell (NSC) Lines"

Article Title: Transcription Activator-Like Effector Nuclease (TALEN)-Mediated CLYBL Targeting Enables Enhanced Transgene Expression and One-Step Generation of Dual Reporter Human Induced Pluripotent Stem Cell (iPSC) and Neural Stem Cell (NSC) Lines

Journal: PLoS ONE

doi: 10.1371/journal.pone.0116032

Effect of targeted integrations on global and local gene expression. (A) Dendrogram of non-targeted and safe-harbor targeted iPSCs and NSCs based on microarray analysis. The X-axis represents 1- r (correlation co-efficient). (GEO access# GSE55975) (B) Representatives of global gene expression profile comparison between untargeted and safe-harbor targeted cells. For NCRM5 iPSCs vs dual safe-harbor targeted NCRM5-AS1Tom-C13GFP clone 1, r =0.991. For NCRM1NSC vs AAVS1 targeted polyclonal NCRM1NSC-AS1-iCLHN. r =0.98. (C) Schematic of genomic region surrounding CLYBL and AAVS1 safe-harbors. TALEN target sites are indicated by red arrows. A ~600kb region near the CLYBL-TALEN target site and a ~300kb region near the AAVS1-TALEN target site are depicted. (D) qRT-PCR analysis of the expression of genes near AAVS1 and CLYBL safe-harbors, as shown in (C), in untargeted NCRM5 iPSCs and dual safe-harbor targeted clone 1. Values are normalized to the expression level of the β-actin (ACTB). ND = not detectable, due to Ct > 35. Native CLYBL and PPP1R12C splicing transcripts were detected by specific primers recognizing exons downstream of TI ( S2 Table ). Fold-expression changes were calculated using the equation 2 -ΔΔCT . Y-axis represents relative quantitation (RQ) in logarithmic scale. The error bars were calculated from 3 technical replicates and display the maximum (RQMax) and minimum (RQMin) expression levels that represent standard error of the mean expression level (RQ value). Collectively, the upper and lower limits defined the region of expression within which the true expression level value was likely to occur. The error bars were based on an RQMin/Max of the 95% confidence level.
Figure Legend Snippet: Effect of targeted integrations on global and local gene expression. (A) Dendrogram of non-targeted and safe-harbor targeted iPSCs and NSCs based on microarray analysis. The X-axis represents 1- r (correlation co-efficient). (GEO access# GSE55975) (B) Representatives of global gene expression profile comparison between untargeted and safe-harbor targeted cells. For NCRM5 iPSCs vs dual safe-harbor targeted NCRM5-AS1Tom-C13GFP clone 1, r =0.991. For NCRM1NSC vs AAVS1 targeted polyclonal NCRM1NSC-AS1-iCLHN. r =0.98. (C) Schematic of genomic region surrounding CLYBL and AAVS1 safe-harbors. TALEN target sites are indicated by red arrows. A ~600kb region near the CLYBL-TALEN target site and a ~300kb region near the AAVS1-TALEN target site are depicted. (D) qRT-PCR analysis of the expression of genes near AAVS1 and CLYBL safe-harbors, as shown in (C), in untargeted NCRM5 iPSCs and dual safe-harbor targeted clone 1. Values are normalized to the expression level of the β-actin (ACTB). ND = not detectable, due to Ct > 35. Native CLYBL and PPP1R12C splicing transcripts were detected by specific primers recognizing exons downstream of TI ( S2 Table ). Fold-expression changes were calculated using the equation 2 -ΔΔCT . Y-axis represents relative quantitation (RQ) in logarithmic scale. The error bars were calculated from 3 technical replicates and display the maximum (RQMax) and minimum (RQMin) expression levels that represent standard error of the mean expression level (RQ value). Collectively, the upper and lower limits defined the region of expression within which the true expression level value was likely to occur. The error bars were based on an RQMin/Max of the 95% confidence level.

Techniques Used: Expressing, Microarray, Quantitative RT-PCR, Quantitation Assay

35) Product Images from "Antiviral innate immunity disturbs podocyte cell function"

Article Title: Antiviral innate immunity disturbs podocyte cell function

Journal: Journal of innate immunity

doi: 10.1159/000345255

TLR3 and RLH signaling molecules in podocytes (A) mRNA was isolated from mouse podocytes (mPod), wild type mouse embryonic fibroblasts (MEFs, +/+), or knock-out mouse embryonic fibroblasts (MEFs, −/−) as indicated in the figure. The expression of each molecule at the mRNA level was tested by RT-PCR. GAPDH bands are representative bands from IRF3 RT-PCR; (B) mRNA from human podocytes (hPod) was tested for each signaling molecule as indicated, by RT-PCR; (C) Expression of TLR3 protein in mouse podocytes (mPod) or mouse embryonic fibroblasts (MEFs) was tested by Western Blot; for both mPod and MEFs, cells from wild type (+/+) mice were compared to cells from mice with targeted deletion of the TLR3 gene (−/−); (D) Expression of TLR3 protein was tested in human podocytes (hPod) by Western Blot. As a positive control (Ctl), lysate from the human fibrosarcoma cell line HT1080 was employed.
Figure Legend Snippet: TLR3 and RLH signaling molecules in podocytes (A) mRNA was isolated from mouse podocytes (mPod), wild type mouse embryonic fibroblasts (MEFs, +/+), or knock-out mouse embryonic fibroblasts (MEFs, −/−) as indicated in the figure. The expression of each molecule at the mRNA level was tested by RT-PCR. GAPDH bands are representative bands from IRF3 RT-PCR; (B) mRNA from human podocytes (hPod) was tested for each signaling molecule as indicated, by RT-PCR; (C) Expression of TLR3 protein in mouse podocytes (mPod) or mouse embryonic fibroblasts (MEFs) was tested by Western Blot; for both mPod and MEFs, cells from wild type (+/+) mice were compared to cells from mice with targeted deletion of the TLR3 gene (−/−); (D) Expression of TLR3 protein was tested in human podocytes (hPod) by Western Blot. As a positive control (Ctl), lysate from the human fibrosarcoma cell line HT1080 was employed.

Techniques Used: Isolation, Knock-Out, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Mouse Assay, Positive Control, CTL Assay

36) Product Images from "Use of an Automated Multiple-Locus, Variable-Number Tandem Repeat-Based Method for Rapid and High-Throughput Genotyping of Staphylococcus aureus Isolates"

Article Title: Use of an Automated Multiple-Locus, Variable-Number Tandem Repeat-Based Method for Rapid and High-Throughput Genotyping of Staphylococcus aureus Isolates

Journal:

doi: 10.1128/JCM.43.7.3346-3355.2005

Effect of lysis duration on DNA yields and PCR amplification results. (A) Amounts of total purified DNA obtained after the rapid lysis procedure (strain MW2), as determined by the fluorescence area under the curve (BioAnalyzer). The electropherogram shown
Figure Legend Snippet: Effect of lysis duration on DNA yields and PCR amplification results. (A) Amounts of total purified DNA obtained after the rapid lysis procedure (strain MW2), as determined by the fluorescence area under the curve (BioAnalyzer). The electropherogram shown

Techniques Used: Lysis, Polymerase Chain Reaction, Amplification, Purification, Fluorescence

37) Product Images from "Messenger RNAs of Yeast Virus-Like Elements Contain Non-templated 5′ Poly(A) Leaders, and Their Expression Is Independent of eIF4E and Pab1"

Article Title: Messenger RNAs of Yeast Virus-Like Elements Contain Non-templated 5′ Poly(A) Leaders, and Their Expression Is Independent of eIF4E and Pab1

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2019.02366

K2ORF5 mRNA is outcompeted by cellular RNA in binding to the yeast cap-binding protein eIF4E in vitro . Electrophoretic analysis of semiquantitative RT-PCR detecting control HGT1 mRNA (upper panel) and K2ORF5 mRNA (lower panel) in samples as follows: Lane 1, glutathione-Sepharose with the bound GST-eIF4E fusion protein in the presence of excess K. lactis IFO1267 total RNA (input); lane 2, same as in line 1 but the reaction was performed without reverse transcriptase (negative control); lane 3, supernatant from the first wash step (unbound mRNA); lanes 4, 5, and 6, supernatants after the second, third, and sixth wash steps, respectively (unbound mRNA); lane 7, mRNA remaining bound on GST-S.c-eIF4E Sepharose after the sixth wash step. M, GeneRuler 100-bp DNA Ladder Plus (Thermo Scientific). PCR was performed using cDNA, Taq DNA polymerase, and the gene-specific primers listed in Supplementary Table S1 . All washing steps were performed with 70 volumes of buffer I. The initial abundances of HGT1 and K2ORF5 mRNA in the K. lactis total RNA were comparable as determined by qRT-PCR ( Supplementary Figure S3 ).
Figure Legend Snippet: K2ORF5 mRNA is outcompeted by cellular RNA in binding to the yeast cap-binding protein eIF4E in vitro . Electrophoretic analysis of semiquantitative RT-PCR detecting control HGT1 mRNA (upper panel) and K2ORF5 mRNA (lower panel) in samples as follows: Lane 1, glutathione-Sepharose with the bound GST-eIF4E fusion protein in the presence of excess K. lactis IFO1267 total RNA (input); lane 2, same as in line 1 but the reaction was performed without reverse transcriptase (negative control); lane 3, supernatant from the first wash step (unbound mRNA); lanes 4, 5, and 6, supernatants after the second, third, and sixth wash steps, respectively (unbound mRNA); lane 7, mRNA remaining bound on GST-S.c-eIF4E Sepharose after the sixth wash step. M, GeneRuler 100-bp DNA Ladder Plus (Thermo Scientific). PCR was performed using cDNA, Taq DNA polymerase, and the gene-specific primers listed in Supplementary Table S1 . All washing steps were performed with 70 volumes of buffer I. The initial abundances of HGT1 and K2ORF5 mRNA in the K. lactis total RNA were comparable as determined by qRT-PCR ( Supplementary Figure S3 ).

Techniques Used: Binding Assay, In Vitro, Reverse Transcription Polymerase Chain Reaction, Negative Control, Polymerase Chain Reaction, Quantitative RT-PCR

Precise manipulation of pGKL VLEs in vivo revealed an essential role of pGKL promoters in mRNA capping and non-template-based 5′ polyadenylation. (A) A closer view of the native pGKL2 region subjected to homologous recombination with a PCR cassette depicted in Supplementary Figure S5 shows a tightly packed VLE genome. The 3′ end of the K2ORF3 coding region overlaps the K2ORF2 promoter, 5′ UTR, and the first four nucleotides of the K2ORF2 coding region. The pGKL2 VLEs displayed in (B,C) are in the reverse orientation of those in Figure 1 . Shades of gray indicate the degree of transcript capping, as shown in Figure 1 . (B) A PCR cassette containing an antibiotic resistance gene (G418) under the control of the ORF2 promoter from pGKL1 ( K1UCR2 ) and the ORF1 promoter from pGKL1 ( K1UCR1 ) ( Supplementary Figure S5 ) was inserted into the K2ORF2 promoter region by homologous recombination in vivo . The resulting VLE, pRKL2-1, contains two genes, aminoglycoside 3′-phosphotransferase (coding for G418 resistance) and K2ORF2 , that are artificially controlled by the pGKL1 promoters K1UCR2 and K1UCR1 , respectively. Shades of gray indicate the degree of transcript capping, as shown in Figure 1 . The 5′ RACE results of pRKL2-1-encoded mRNAs are summarized in the text and in Supplementary Table S7 . (C) Electrophoretic analysis of pGKL VLEs in K. lactis clones. M, lambda DNA/ Eco 130I ( Sty I) marker (Fermentas); lanes 1 and 4, native pGKL VLEs from K. lactis IFO1267 (pGKL1 [8874 bp] is labeled with an asterisk, and pGKL2 [13447 bp] is labeled with an arrow); lane 2, linear VLEs purified from K. lactis IFO1267 carrying both the recombinant (higher MW) and wild-type pGKL2 VLEs; lane 3, linear VLEs purified from K. lactis IFO1267 containing the recombinant pRKL2-1 VLE (14353 bp). The shorter wild-type pGKL2 was lost after cultivation for ≈60 generations in selective medium containing G418.
Figure Legend Snippet: Precise manipulation of pGKL VLEs in vivo revealed an essential role of pGKL promoters in mRNA capping and non-template-based 5′ polyadenylation. (A) A closer view of the native pGKL2 region subjected to homologous recombination with a PCR cassette depicted in Supplementary Figure S5 shows a tightly packed VLE genome. The 3′ end of the K2ORF3 coding region overlaps the K2ORF2 promoter, 5′ UTR, and the first four nucleotides of the K2ORF2 coding region. The pGKL2 VLEs displayed in (B,C) are in the reverse orientation of those in Figure 1 . Shades of gray indicate the degree of transcript capping, as shown in Figure 1 . (B) A PCR cassette containing an antibiotic resistance gene (G418) under the control of the ORF2 promoter from pGKL1 ( K1UCR2 ) and the ORF1 promoter from pGKL1 ( K1UCR1 ) ( Supplementary Figure S5 ) was inserted into the K2ORF2 promoter region by homologous recombination in vivo . The resulting VLE, pRKL2-1, contains two genes, aminoglycoside 3′-phosphotransferase (coding for G418 resistance) and K2ORF2 , that are artificially controlled by the pGKL1 promoters K1UCR2 and K1UCR1 , respectively. Shades of gray indicate the degree of transcript capping, as shown in Figure 1 . The 5′ RACE results of pRKL2-1-encoded mRNAs are summarized in the text and in Supplementary Table S7 . (C) Electrophoretic analysis of pGKL VLEs in K. lactis clones. M, lambda DNA/ Eco 130I ( Sty I) marker (Fermentas); lanes 1 and 4, native pGKL VLEs from K. lactis IFO1267 (pGKL1 [8874 bp] is labeled with an asterisk, and pGKL2 [13447 bp] is labeled with an arrow); lane 2, linear VLEs purified from K. lactis IFO1267 carrying both the recombinant (higher MW) and wild-type pGKL2 VLEs; lane 3, linear VLEs purified from K. lactis IFO1267 containing the recombinant pRKL2-1 VLE (14353 bp). The shorter wild-type pGKL2 was lost after cultivation for ≈60 generations in selective medium containing G418.

Techniques Used: In Vivo, Homologous Recombination, Polymerase Chain Reaction, Clone Assay, Lambda DNA Preparation, Marker, Labeling, Purification, Recombinant

Differences in the lengths of the 5′ mRNA poly(A) leaders of individual pGKL ORFs. The box whisker plot represents the number of templated and non-templated consecutive adenosine nucleotides at the 5′ ends of pGKL mRNAs. The bottom and top of the box indicate the first and third quartiles, respectively. The whiskers indicate the 10th and 90th percentiles. The median is indicated as a solid black line. Outliers are not indicated. The open reading frames are ranked from left to right according to the prevalent lengths of the 5′ poly(A) leaders of their mRNAs. The value of 12 adenosine nucleotides represents an optimal length of the Pab1 binding site and is indicated as a solid gray line. The pGKL1 mRNAs belong to those with the longest 5′ poly(A) leaders. The data did not follow a normal distribution according to the Shapiro–Wilk test. The results were statistically analyzed using the non-parametric Kruskal–Wallis test, which supported rejection of the null hypothesis, p -value = 2.172940e-48. To discern gene pairs, the transcripts of which significantly differed in the lengths of their 5′ poly(A) leaders, we performed Dunn post hoc tests followed by adjustment of p -values according to the Benjamini–Hochberg FDR method. Adjusted p -values corresponding to all possible gene pairs are depicted in Supplementary Table S10 . In total, 458 sequences obtained by 5′ RACE-PCR were used for this analysis ( Supplementary Table S5A ).
Figure Legend Snippet: Differences in the lengths of the 5′ mRNA poly(A) leaders of individual pGKL ORFs. The box whisker plot represents the number of templated and non-templated consecutive adenosine nucleotides at the 5′ ends of pGKL mRNAs. The bottom and top of the box indicate the first and third quartiles, respectively. The whiskers indicate the 10th and 90th percentiles. The median is indicated as a solid black line. Outliers are not indicated. The open reading frames are ranked from left to right according to the prevalent lengths of the 5′ poly(A) leaders of their mRNAs. The value of 12 adenosine nucleotides represents an optimal length of the Pab1 binding site and is indicated as a solid gray line. The pGKL1 mRNAs belong to those with the longest 5′ poly(A) leaders. The data did not follow a normal distribution according to the Shapiro–Wilk test. The results were statistically analyzed using the non-parametric Kruskal–Wallis test, which supported rejection of the null hypothesis, p -value = 2.172940e-48. To discern gene pairs, the transcripts of which significantly differed in the lengths of their 5′ poly(A) leaders, we performed Dunn post hoc tests followed by adjustment of p -values according to the Benjamini–Hochberg FDR method. Adjusted p -values corresponding to all possible gene pairs are depicted in Supplementary Table S10 . In total, 458 sequences obtained by 5′ RACE-PCR were used for this analysis ( Supplementary Table S5A ).

Techniques Used: Whisker Assay, Binding Assay, Polymerase Chain Reaction

38) Product Images from "Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance"

Article Title: Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance

Journal: PLoS ONE

doi: 10.1371/journal.pone.0036494

Schematic representation of the sample preparation for ultra-wide HIV drug resistance genotyping using Roche/454 pyrosequencing. A.) Plasma is isolated from 48 patient samples using centrifugation. B.) Viral RNA is extracted from ∼1 ml of plasma from each sample. C.) One-step RT-PCR is used to reverse-transcribe and PCR-amplify 3 amplicons spanning the HIV pol gene from each sample as shown. Each sample is amplified with primers containing a unique multiplex identifier (MID) tag (1–48). D.) PCR products are gel purified and purified further using size exclusion magnetic beads. E.) Purified samples are quantitated and pooled together at equimolar ratios for a total of 144 amplicons/pool. F.) Each pool is subjected to emPCR followed by pyrosequencing on the Roche/454 GS Junior.
Figure Legend Snippet: Schematic representation of the sample preparation for ultra-wide HIV drug resistance genotyping using Roche/454 pyrosequencing. A.) Plasma is isolated from 48 patient samples using centrifugation. B.) Viral RNA is extracted from ∼1 ml of plasma from each sample. C.) One-step RT-PCR is used to reverse-transcribe and PCR-amplify 3 amplicons spanning the HIV pol gene from each sample as shown. Each sample is amplified with primers containing a unique multiplex identifier (MID) tag (1–48). D.) PCR products are gel purified and purified further using size exclusion magnetic beads. E.) Purified samples are quantitated and pooled together at equimolar ratios for a total of 144 amplicons/pool. F.) Each pool is subjected to emPCR followed by pyrosequencing on the Roche/454 GS Junior.

Techniques Used: Sample Prep, Isolation, Centrifugation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Multiplex Assay, Purification, Magnetic Beads

Sequence coverage of three amplicons from a clonal HXB2 viral stock and HXB2 plasmid. The number of sequences from the clonal viral stock (red) or HIV plasmid (blue and black) that aligned to each nucleotide position of the NC_001802 HXB2 HIV reference sequence is shown across the pol gene. The HXB2 vRNA clonal viral stock was sequenced under one GS Junior sequencing run, while two independent PCR amplifications were used to sequence the plasmid under two different MID tags in a separate GS Junior sequencing run. Pyrosequencing was performed on three overlapping amplicons with nucleotide positions for each amplicon represented at the top of the graph.
Figure Legend Snippet: Sequence coverage of three amplicons from a clonal HXB2 viral stock and HXB2 plasmid. The number of sequences from the clonal viral stock (red) or HIV plasmid (blue and black) that aligned to each nucleotide position of the NC_001802 HXB2 HIV reference sequence is shown across the pol gene. The HXB2 vRNA clonal viral stock was sequenced under one GS Junior sequencing run, while two independent PCR amplifications were used to sequence the plasmid under two different MID tags in a separate GS Junior sequencing run. Pyrosequencing was performed on three overlapping amplicons with nucleotide positions for each amplicon represented at the top of the graph.

Techniques Used: Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Amplification

39) Product Images from "Excessive folic acid supplementation in pregnant mice impairs insulin secretion and induces the expression of genes associated with fatty liver in their offspring"

Article Title: Excessive folic acid supplementation in pregnant mice impairs insulin secretion and induces the expression of genes associated with fatty liver in their offspring

Journal: Heliyon

doi: 10.1016/j.heliyon.2020.e03597

mRNA expression of genes involved in carbohydrate metabolism. Analysis of the mRNA expression of genes involved in carbohydrate metabolism was performed in offspring liver using real-time RT-PCR. A) Pparγ1 , B) Pparγ2 , C) Acaca , D) Acacb , E) C idec , F) Acox1 , G) Fasn , H) Gpd1 , I) Pck1 , J) Pck2 , K) Ggat1 , and L) G gat2 . Control group (CN; white bars); high-folic acid group (HFA; black bars), The fold difference between the HFA and CN groups are shown at the top of the graphs (∗, P
Figure Legend Snippet: mRNA expression of genes involved in carbohydrate metabolism. Analysis of the mRNA expression of genes involved in carbohydrate metabolism was performed in offspring liver using real-time RT-PCR. A) Pparγ1 , B) Pparγ2 , C) Acaca , D) Acacb , E) C idec , F) Acox1 , G) Fasn , H) Gpd1 , I) Pck1 , J) Pck2 , K) Ggat1 , and L) G gat2 . Control group (CN; white bars); high-folic acid group (HFA; black bars), The fold difference between the HFA and CN groups are shown at the top of the graphs (∗, P

Techniques Used: Expressing, Quantitative RT-PCR

40) Product Images from "A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo virulence and transmissibility properties of the parental virus"

Article Title: A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo virulence and transmissibility properties of the parental virus

Journal: Virology

doi: 10.1016/j.virol.2004.04.046

The cloned virus retains in vivo markers of virulence and is genetically stable. Three groups of pigs (four pigs per group) were sham-inoculated or inoculated with either vParental or vFL12 at TCID 50 /ml of 10 5.2 and 10 4.8 , respectively. (A) Temperatures were recorded daily from 2 days before inoculation to 16 dpi. Average temperatures from four different animals in each group are shown. (B) Serum samples were collected at 4, 7, 14, and 22 dpi and virus titers were determined and expressed as TCID 50 /ml. Values represent average titers from four animals. (C) Serum antibody titers were determined with the Idexx ELISA kit. Values represent average titers from four animals. Dashed line at 0.4 S/P ratio designates threshold value above which titers are considered positive for anti-PRRSV antibodies. Error bars in panels A, B, and C represent standard deviation. (D) Serum from 14 dpi, recovered from inoculated animals, was used to distinguish between vFL12 and vParental by examining for the presence of the Bsr GI genetic tag (left panel). Viral RNA extracted from sera was amplified by RT-PCR and the products were digested with Bsr GI. Similar analysis (lane 7) was performed with virus in serum from a sentinel pig at 8 days postcohabitation with pigs infected with vFL12 (right panel). Gel image is inverted. Error bars represent standard deviation.
Figure Legend Snippet: The cloned virus retains in vivo markers of virulence and is genetically stable. Three groups of pigs (four pigs per group) were sham-inoculated or inoculated with either vParental or vFL12 at TCID 50 /ml of 10 5.2 and 10 4.8 , respectively. (A) Temperatures were recorded daily from 2 days before inoculation to 16 dpi. Average temperatures from four different animals in each group are shown. (B) Serum samples were collected at 4, 7, 14, and 22 dpi and virus titers were determined and expressed as TCID 50 /ml. Values represent average titers from four animals. (C) Serum antibody titers were determined with the Idexx ELISA kit. Values represent average titers from four animals. Dashed line at 0.4 S/P ratio designates threshold value above which titers are considered positive for anti-PRRSV antibodies. Error bars in panels A, B, and C represent standard deviation. (D) Serum from 14 dpi, recovered from inoculated animals, was used to distinguish between vFL12 and vParental by examining for the presence of the Bsr GI genetic tag (left panel). Viral RNA extracted from sera was amplified by RT-PCR and the products were digested with Bsr GI. Similar analysis (lane 7) was performed with virus in serum from a sentinel pig at 8 days postcohabitation with pigs infected with vFL12 (right panel). Gel image is inverted. Error bars represent standard deviation.

Techniques Used: Clone Assay, In Vivo, Enzyme-linked Immunosorbent Assay, Standard Deviation, Amplification, Reverse Transcription Polymerase Chain Reaction, Infection

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SYBR Green Assay:

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Polymerase Chain Reaction:

Article Title: Detection of methicillin-resistance gene in Staphylococcus epidermidis strains isolated from patients in Al-Zahra Hospital using polymerase chain reaction and minimum inhibitory concentration methods
Article Snippet: .. The following primers were used for PCR amplification of mecA gene:[ ] mecA-F: 5′-TGGCTATCGTGTCACAATCG-3′ mecA-R: 5′-CTGGAACTTGTTGAGCAGAG-3′ PCR was performed in a mixture of 25 μl volume containing: 2.5 μl 10 × buffer (Roche Germany, Berlin), 0.4 μl of each dNTP (200 μm), 2.5 μl (50 mm) MgCl2, 2.5 U of Taq DNA polymerase, 10 pmol of each primer, and 5 μl of template DNA. ..

Article Title: Expression of HOXA11 in the mid-luteal endometrium from women with endometriosis-associated infertility
Article Snippet: .. PCR amplification was conducted by FastStart Taq DNA Polymerase from Roche Diagnostic GmbH (Mannheim, Germany). .. The PCR products were purified using Agarose Gel DNA Extraction Kit Roche (Mannheim, Germany) with subsequent cloning into pGEM-T Easy Vector System I Promega (Madison, WI) and transformation into TOPO10 E. coli strain cells.

Article Title: Nodulin 41, a novel late nodulin of common bean with peptidase activity
Article Snippet: .. Amino acid sequencing, PCR amplification and cloning of PvNod41 100 μg of pure PvNod41 were digested with 5 μg of trypsin (sequencing grade; Roche, Mannheim, Germany) in 50 mM Tris-HCl pH 8.0 and the resulting peptides were purified by reversed-phase HPLC by using a C-18 analytical column (Vydac, Hesperia, CA, USA). ..

Article Title: Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer
Article Snippet: .. PCR amplification was performed by FastStart Taq DNA Polymerase from Roche Diagnostic GmbH (Mannheim, Germany). .. The PCR products were purified using Agarose Gel DNA Extraction Kit, Roche Diagnostic GmbH (Mannheim, Germany) with subsequent cloning into pGEM-T Easy Vector System I, Promega (Madison, WI) and transformation into TOPO10 E. coli strain cells.

Article Title: Up-regulation of C5a receptor expression and function on human monocyte derived dendritic cells by prostaglandin E2
Article Snippet: .. The following primers were used for PCR amplification: C5aR sense: 5′-GAG CCC AGG AGA CCA GAA CAT G, and C5aR antisense: 5′-TAC ATG TTG AGC AGG ATG AGG GA, β-actin sense: 5′-AAG GCC AAC CGC GAG AAG ATG A, and β-actin antisense: 5′-GGA AGA GTG CCT CAG GGC AGC G. PCR was performed on a LightCycler (Roche Molecular Biochemicals) in LightCycler capillaries using a commercially available master mix containing Taq DNA polymerase, SYBR-Green I, dNTPs (LightCycler DNA master SYBR-Green I, Roche Molecular Biochemicals). .. After addition of primers (final concentration: 0·25 p m ), MgCl2 (3·5 m m ) and template DNA to the master mix, 37 cycles of denaturation (94° for 1 s), annealing (55° for 5 s) and extension (72° for 12 s) were performed.

Article Title: The NADPH oxidase Nox4 restricts the replicative lifespan of human endothelial cells
Article Snippet: .. The cDNA equivalent of 5 ng of RNA was applied to PCR amplification in combination with 15 μl of LightCycler® 480 SYBR Green I Master (Roche Applied Science), a reaction mixture including FastStart Taq DNA Polymerase and SYBR Green I dye for product detection. cDNA concentrations were normalized by the use of the PBGD internal standard. .. Real-time PCR was performed in triplicate in the LightCycler® 480 Instrument (Roche Applied Science) in a final reaction volume of 50 μl per tube.

Article Title: A DNA Vaccine against Yellow Fever Virus: Development and Evaluation
Article Snippet: .. PCR amplification was performed using the proofreading TGO DNA polymerase (Roche, Indianapolis, IN, USA) and 0.6 μM of each primer. .. The amplicon was inserted into the p43.2 vector between the Xho I and Not I cleavage sites to generate the p/YFE construct.

Article Title: Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates
Article Snippet: .. We thus assessed the effect of exonuclease III on long PCR amplification performed with the Expand™ Long Template PCR system (Roche Molecular Biochemicals), which combines the Taq and Pwo DNA polymerases. .. We found that exonuclease III enhanced the amplification of different types of damaged DNA templates when PCR reactions were performed with this combination of DNA polymerases (data not shown).

Sequencing:

Article Title: Nodulin 41, a novel late nodulin of common bean with peptidase activity
Article Snippet: .. Amino acid sequencing, PCR amplification and cloning of PvNod41 100 μg of pure PvNod41 were digested with 5 μg of trypsin (sequencing grade; Roche, Mannheim, Germany) in 50 mM Tris-HCl pH 8.0 and the resulting peptides were purified by reversed-phase HPLC by using a C-18 analytical column (Vydac, Hesperia, CA, USA). ..

Chloramphenicol Acetyltransferase Assay:

Article Title: Up-regulation of C5a receptor expression and function on human monocyte derived dendritic cells by prostaglandin E2
Article Snippet: .. The following primers were used for PCR amplification: C5aR sense: 5′-GAG CCC AGG AGA CCA GAA CAT G, and C5aR antisense: 5′-TAC ATG TTG AGC AGG ATG AGG GA, β-actin sense: 5′-AAG GCC AAC CGC GAG AAG ATG A, and β-actin antisense: 5′-GGA AGA GTG CCT CAG GGC AGC G. PCR was performed on a LightCycler (Roche Molecular Biochemicals) in LightCycler capillaries using a commercially available master mix containing Taq DNA polymerase, SYBR-Green I, dNTPs (LightCycler DNA master SYBR-Green I, Roche Molecular Biochemicals). .. After addition of primers (final concentration: 0·25 p m ), MgCl2 (3·5 m m ) and template DNA to the master mix, 37 cycles of denaturation (94° for 1 s), annealing (55° for 5 s) and extension (72° for 12 s) were performed.

Countercurrent Chromatography:

Article Title: Up-regulation of C5a receptor expression and function on human monocyte derived dendritic cells by prostaglandin E2
Article Snippet: .. The following primers were used for PCR amplification: C5aR sense: 5′-GAG CCC AGG AGA CCA GAA CAT G, and C5aR antisense: 5′-TAC ATG TTG AGC AGG ATG AGG GA, β-actin sense: 5′-AAG GCC AAC CGC GAG AAG ATG A, and β-actin antisense: 5′-GGA AGA GTG CCT CAG GGC AGC G. PCR was performed on a LightCycler (Roche Molecular Biochemicals) in LightCycler capillaries using a commercially available master mix containing Taq DNA polymerase, SYBR-Green I, dNTPs (LightCycler DNA master SYBR-Green I, Roche Molecular Biochemicals). .. After addition of primers (final concentration: 0·25 p m ), MgCl2 (3·5 m m ) and template DNA to the master mix, 37 cycles of denaturation (94° for 1 s), annealing (55° for 5 s) and extension (72° for 12 s) were performed.

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  • 85
    Roche negative strand hcv pcr amplifications
    Effect of increasing concentrations of IFN-α on the accumulation of positive- and negative-strand <t>HCV</t> RNA in primary hepatocyte cultures infected in vitro. Cultures FT147 (infected with serum S26) and FT161 (infected with serum S42) were treated for 5 and 8 days with IFN-α concentrations ranging from 1,000 to 10,000 U/ml and 500 to 5,000 U/ml, respectively. Qualitative detection of positive-sense (+) and negative-sense (-) HCV RNA strands is shown in cultures FT147 (a) and FT161 (b). In both instances, positive-strand HCV RNA was detected at all concentrations used, whereas the negative strand was never detected. MW, molecular size standards. (c) In culture FT161, LightCycler real-time <t>RT-PCR</t> quantitative analysis of the same extracts showed a reduction in the amount of positive-sense HCV RNA strand in the culture when the IFN-α concentration increased, suggesting IFN-α concentration-dependent inhibition of HCV replication in the culture. Similar results (not shown) were obtained with culture FT187 infected with serum S155.
    Negative Strand Hcv Pcr Amplifications, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche qrt pcr amplification protocol
    NBAT1 inhibits invasion of breast cancer cells by activating DKK1 expression a. , b . <t>qRT-PCR</t> and western blot analysis for DKK1 in NBAT1-expression MDA-MB-231 cells transfected with siRNA targeting DKK1 (NC, siDKK1-1 and siDKK1-2). c . Representative images of Boyden chamber assay for invaded cells (over-expression NBAT1 while inhibit DKK1). d . Histogram showing that the number of invaded cells with knockdown DKK1 was significantly higher than for NC, and similar to control groups (untreated, mock and vector, mean±SD, n=3, * P
    Qrt Pcr Amplification Protocol, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qrt pcr amplification protocol/product/Roche
    Average 88 stars, based on 1 article reviews
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    85
    Roche real time quantitative pcr qpcr amplifications
    Relative expression of Cg-BigDef transcripts in oyster hemocytes by real-time quantitative <t>PCR.</t> a : Expression analysis of the three Cg-BigDef forms at 12 h post-stimulation with sterile sea water (white bars, SSW) and heat-killed bacteria (black bars, stimulation). Asterisks (*) indicate significant differences between conditions according to the Student's t-test ( p
    Real Time Quantitative Pcr Qpcr Amplifications, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche qrt pcr amplification
    Validation of RNA-Seq data using <t>qRT-PCR.</t> Correlation of expression levels of 12 selected DEGs derived from log 2 [fold change] in G_GG, G_GB, B_BB, and B_BG pairwise comparisons were determined by linear fitting the RNA-Seq and qRT-PCR data. Error bars represent the SD ( n = 3).
    Qrt Pcr Amplification, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of increasing concentrations of IFN-α on the accumulation of positive- and negative-strand HCV RNA in primary hepatocyte cultures infected in vitro. Cultures FT147 (infected with serum S26) and FT161 (infected with serum S42) were treated for 5 and 8 days with IFN-α concentrations ranging from 1,000 to 10,000 U/ml and 500 to 5,000 U/ml, respectively. Qualitative detection of positive-sense (+) and negative-sense (-) HCV RNA strands is shown in cultures FT147 (a) and FT161 (b). In both instances, positive-strand HCV RNA was detected at all concentrations used, whereas the negative strand was never detected. MW, molecular size standards. (c) In culture FT161, LightCycler real-time RT-PCR quantitative analysis of the same extracts showed a reduction in the amount of positive-sense HCV RNA strand in the culture when the IFN-α concentration increased, suggesting IFN-α concentration-dependent inhibition of HCV replication in the culture. Similar results (not shown) were obtained with culture FT187 infected with serum S155.

    Journal: Journal of Virology

    Article Title: Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro

    doi: 10.1128/JVI.76.16.8189-8199.2002

    Figure Lengend Snippet: Effect of increasing concentrations of IFN-α on the accumulation of positive- and negative-strand HCV RNA in primary hepatocyte cultures infected in vitro. Cultures FT147 (infected with serum S26) and FT161 (infected with serum S42) were treated for 5 and 8 days with IFN-α concentrations ranging from 1,000 to 10,000 U/ml and 500 to 5,000 U/ml, respectively. Qualitative detection of positive-sense (+) and negative-sense (-) HCV RNA strands is shown in cultures FT147 (a) and FT161 (b). In both instances, positive-strand HCV RNA was detected at all concentrations used, whereas the negative strand was never detected. MW, molecular size standards. (c) In culture FT161, LightCycler real-time RT-PCR quantitative analysis of the same extracts showed a reduction in the amount of positive-sense HCV RNA strand in the culture when the IFN-α concentration increased, suggesting IFN-α concentration-dependent inhibition of HCV replication in the culture. Similar results (not shown) were obtained with culture FT187 infected with serum S155.

    Article Snippet: Positive- and negative-strand HCV PCR amplifications were performed with 3 μl of purified cDNA in a 10-μl reaction mixture containing 1 μl of LightCycler-FastStart DNA Master SYBR green (Roche Applied Science) and 0.5 μM (each) HCV primer KY78 and KY80.

    Techniques: Infection, In Vitro, Quantitative RT-PCR, Concentration Assay, Inhibition

    Qualitative assay detection of positive- and negative-strand HCV RNA in a primary culture of healthy human hepatocytes infected in vitro with an HCV-positive serum and effect of IFN-α. The hepatocyte culture FT147, infected 3 days after plating by HCV-positive serum S26, is shown. Positive-strand (+) RNA but not negative-strand (-) RNA was present in the inoculum. (a) Primary hepatocyte culture in the absence of IFN-α. Positive-strand HCV RNA was detected with the qualitative strand-specific r Tth PCR assay from day 1 to the end of the culture (day 12), whereas negative-strand RNA was detected from days 2 to 10. (b) Culture in the presence of 5,000 U of IFN-α per ml. Positive-strand HCV RNA was detected from days 1 to 10, whereas negative-strand RNA was never detected. (c) Culture treated on day 3 with 5,000 U of IFN-α per ml. Positive-strand RNA was detected throughout the culture period, whereas negative-strand RNA was no longer detected after day 5. Similar patterns (not shown) were observed with the following cultures infected with the corresponding sera: FT141 and S23, FT143 and S34, FT144 and S27, FT154 and S23, FT155 and S20, and FT156 and S17. MK, molecular size standards.

    Journal: Journal of Virology

    Article Title: Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro

    doi: 10.1128/JVI.76.16.8189-8199.2002

    Figure Lengend Snippet: Qualitative assay detection of positive- and negative-strand HCV RNA in a primary culture of healthy human hepatocytes infected in vitro with an HCV-positive serum and effect of IFN-α. The hepatocyte culture FT147, infected 3 days after plating by HCV-positive serum S26, is shown. Positive-strand (+) RNA but not negative-strand (-) RNA was present in the inoculum. (a) Primary hepatocyte culture in the absence of IFN-α. Positive-strand HCV RNA was detected with the qualitative strand-specific r Tth PCR assay from day 1 to the end of the culture (day 12), whereas negative-strand RNA was detected from days 2 to 10. (b) Culture in the presence of 5,000 U of IFN-α per ml. Positive-strand HCV RNA was detected from days 1 to 10, whereas negative-strand RNA was never detected. (c) Culture treated on day 3 with 5,000 U of IFN-α per ml. Positive-strand RNA was detected throughout the culture period, whereas negative-strand RNA was no longer detected after day 5. Similar patterns (not shown) were observed with the following cultures infected with the corresponding sera: FT141 and S23, FT143 and S34, FT144 and S27, FT154 and S23, FT155 and S20, and FT156 and S17. MK, molecular size standards.

    Article Snippet: Positive- and negative-strand HCV PCR amplifications were performed with 3 μl of purified cDNA in a 10-μl reaction mixture containing 1 μl of LightCycler-FastStart DNA Master SYBR green (Roche Applied Science) and 0.5 μM (each) HCV primer KY78 and KY80.

    Techniques: Infection, In Vitro, Polymerase Chain Reaction

    Characteristics of the strand-specific HCV RNA assays used in this study. (a) Strand specificity of the positive-strand-specific HCV RNA r Tth RT-PCR assay. Decreasing amounts of positive-strand (+) HCV RNA (100, 10, 1, and 0.1 fg) and of negative-strand (-) HCV RNA (10, 1, and 0.1 pg) synthesized from an appropriate plasmid were subjected to the r Tth RT-PCR assay. The products were analyzed by agarose gel electrophoresis. (b) Strand specificity of the negative-strand-specific HCV RNA r Tth RT-PCR assay. Decreasing amounts of negative-strand HCV RNA (100, 10, 1, and 0.1 fg) and positive-strand HCV RNA (10, 1, and 0.1 pg) synthesized from the same plasmid as for panel a were analyzed by the same procedure. (c) Range of linear quantification of the quantitative assay based on real-time PCR using the LightCycler technology and SYBR green I dye for detection. The range of linear quantification of the assay was studied by testing 10-fold serial dilutions of synthetic positive- and negative-sense HCV RNA strands after RT at 70°C with the r Tth polymerase. Each point is the mean of three experimental values for each dilution. y is the slope of the linear plots.

    Journal: Journal of Virology

    Article Title: Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro

    doi: 10.1128/JVI.76.16.8189-8199.2002

    Figure Lengend Snippet: Characteristics of the strand-specific HCV RNA assays used in this study. (a) Strand specificity of the positive-strand-specific HCV RNA r Tth RT-PCR assay. Decreasing amounts of positive-strand (+) HCV RNA (100, 10, 1, and 0.1 fg) and of negative-strand (-) HCV RNA (10, 1, and 0.1 pg) synthesized from an appropriate plasmid were subjected to the r Tth RT-PCR assay. The products were analyzed by agarose gel electrophoresis. (b) Strand specificity of the negative-strand-specific HCV RNA r Tth RT-PCR assay. Decreasing amounts of negative-strand HCV RNA (100, 10, 1, and 0.1 fg) and positive-strand HCV RNA (10, 1, and 0.1 pg) synthesized from the same plasmid as for panel a were analyzed by the same procedure. (c) Range of linear quantification of the quantitative assay based on real-time PCR using the LightCycler technology and SYBR green I dye for detection. The range of linear quantification of the assay was studied by testing 10-fold serial dilutions of synthetic positive- and negative-sense HCV RNA strands after RT at 70°C with the r Tth polymerase. Each point is the mean of three experimental values for each dilution. y is the slope of the linear plots.

    Article Snippet: Positive- and negative-strand HCV PCR amplifications were performed with 3 μl of purified cDNA in a 10-μl reaction mixture containing 1 μl of LightCycler-FastStart DNA Master SYBR green (Roche Applied Science) and 0.5 μM (each) HCV primer KY78 and KY80.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Synthesized, Plasmid Preparation, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, SYBR Green Assay

    Accumulation of positive- and negative-strand HCV RNA in hepatocyte cultures FT172 (a), FT189 (b), and FT195 (c), infected with sera S42, S155, and S155, respectively, as measured by the quantitative LightCycler real-time RT-PCR assay. The hepatocyte cultures were infected 3 days after plating. The cells were harvested 30 min and 1, 3, and 5 days after infection for positive-strand (gray) and negative-strand (black) HCV RNA quantification. The amounts of HCV RNA strands are shown as means ± SEMs of three determinations, expressed in numbers of HCV RNA copies per 2 × 10 6 cells, normalized to GAPDH mRNA. Similar results (not shown) were obtained with culture FT168 infected with serum S34.

    Journal: Journal of Virology

    Article Title: Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro

    doi: 10.1128/JVI.76.16.8189-8199.2002

    Figure Lengend Snippet: Accumulation of positive- and negative-strand HCV RNA in hepatocyte cultures FT172 (a), FT189 (b), and FT195 (c), infected with sera S42, S155, and S155, respectively, as measured by the quantitative LightCycler real-time RT-PCR assay. The hepatocyte cultures were infected 3 days after plating. The cells were harvested 30 min and 1, 3, and 5 days after infection for positive-strand (gray) and negative-strand (black) HCV RNA quantification. The amounts of HCV RNA strands are shown as means ± SEMs of three determinations, expressed in numbers of HCV RNA copies per 2 × 10 6 cells, normalized to GAPDH mRNA. Similar results (not shown) were obtained with culture FT168 infected with serum S34.

    Article Snippet: Positive- and negative-strand HCV PCR amplifications were performed with 3 μl of purified cDNA in a 10-μl reaction mixture containing 1 μl of LightCycler-FastStart DNA Master SYBR green (Roche Applied Science) and 0.5 μM (each) HCV primer KY78 and KY80.

    Techniques: Infection, Quantitative RT-PCR

    NBAT1 inhibits invasion of breast cancer cells by activating DKK1 expression a. , b . qRT-PCR and western blot analysis for DKK1 in NBAT1-expression MDA-MB-231 cells transfected with siRNA targeting DKK1 (NC, siDKK1-1 and siDKK1-2). c . Representative images of Boyden chamber assay for invaded cells (over-expression NBAT1 while inhibit DKK1). d . Histogram showing that the number of invaded cells with knockdown DKK1 was significantly higher than for NC, and similar to control groups (untreated, mock and vector, mean±SD, n=3, * P

    Journal: Oncotarget

    Article Title: NBAT1 suppresses breast cancer metastasis by regulating DKK1 via PRC2

    doi:

    Figure Lengend Snippet: NBAT1 inhibits invasion of breast cancer cells by activating DKK1 expression a. , b . qRT-PCR and western blot analysis for DKK1 in NBAT1-expression MDA-MB-231 cells transfected with siRNA targeting DKK1 (NC, siDKK1-1 and siDKK1-2). c . Representative images of Boyden chamber assay for invaded cells (over-expression NBAT1 while inhibit DKK1). d . Histogram showing that the number of invaded cells with knockdown DKK1 was significantly higher than for NC, and similar to control groups (untreated, mock and vector, mean±SD, n=3, * P

    Article Snippet: The qRT-PCR amplification protocol was conducted according to the user guide of the SYBR® Green master mixes on the Roche LightCycler® 480 Real-Time PCR System.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Multiple Displacement Amplification, Transfection, Boyden Chamber Assay, Over Expression, Plasmid Preparation

    Over-expression of NBAT1 in MDA-MB-231 cells results in global gene expression profile change a . Heatmap representing hierarchical clustering of all dysregulated genes whose relative fold changes are more than 2 times compared MDA-MB-231/NBAT1 with MDA-MB-231/vector cells. b . Pathway-network analysis of the significant pathways of the differential expression genes. (Lines represent the relationship between the pathways, red to white represents the P value; the smaller the P value is, the deeper the red is.) c . The expression levels of DKK1, PRLR, NUPR1, PTGS2, WNT11 were determined in MDA-MB-231 with over-expression NBAT1 by qRT-PCR (mean±SD, n=3, *** p

    Journal: Oncotarget

    Article Title: NBAT1 suppresses breast cancer metastasis by regulating DKK1 via PRC2

    doi:

    Figure Lengend Snippet: Over-expression of NBAT1 in MDA-MB-231 cells results in global gene expression profile change a . Heatmap representing hierarchical clustering of all dysregulated genes whose relative fold changes are more than 2 times compared MDA-MB-231/NBAT1 with MDA-MB-231/vector cells. b . Pathway-network analysis of the significant pathways of the differential expression genes. (Lines represent the relationship between the pathways, red to white represents the P value; the smaller the P value is, the deeper the red is.) c . The expression levels of DKK1, PRLR, NUPR1, PTGS2, WNT11 were determined in MDA-MB-231 with over-expression NBAT1 by qRT-PCR (mean±SD, n=3, *** p

    Article Snippet: The qRT-PCR amplification protocol was conducted according to the user guide of the SYBR® Green master mixes on the Roche LightCycler® 480 Real-Time PCR System.

    Techniques: Over Expression, Multiple Displacement Amplification, Expressing, Plasmid Preparation, Quantitative RT-PCR

    NBAT1 inhibits invasion of breast cancer cells via EZH2 a . Binding of NBAT1 to EZH2 complex in MDA-MB-231 cells, shown by RNA immunoprecipitation followed qRT-PCR (mean±SD, n=3, *** p

    Journal: Oncotarget

    Article Title: NBAT1 suppresses breast cancer metastasis by regulating DKK1 via PRC2

    doi:

    Figure Lengend Snippet: NBAT1 inhibits invasion of breast cancer cells via EZH2 a . Binding of NBAT1 to EZH2 complex in MDA-MB-231 cells, shown by RNA immunoprecipitation followed qRT-PCR (mean±SD, n=3, *** p

    Article Snippet: The qRT-PCR amplification protocol was conducted according to the user guide of the SYBR® Green master mixes on the Roche LightCycler® 480 Real-Time PCR System.

    Techniques: Binding Assay, Multiple Displacement Amplification, Immunoprecipitation, Quantitative RT-PCR

    Relative expression of Cg-BigDef transcripts in oyster hemocytes by real-time quantitative PCR. a : Expression analysis of the three Cg-BigDef forms at 12 h post-stimulation with sterile sea water (white bars, SSW) and heat-killed bacteria (black bars, stimulation). Asterisks (*) indicate significant differences between conditions according to the Student's t-test ( p

    Journal: PLoS ONE

    Article Title: Big Defensins, a Diverse Family of Antimicrobial Peptides That Follows Different Patterns of Expression in Hemocytes of the Oyster Crassostrea gigas

    doi: 10.1371/journal.pone.0025594

    Figure Lengend Snippet: Relative expression of Cg-BigDef transcripts in oyster hemocytes by real-time quantitative PCR. a : Expression analysis of the three Cg-BigDef forms at 12 h post-stimulation with sterile sea water (white bars, SSW) and heat-killed bacteria (black bars, stimulation). Asterisks (*) indicate significant differences between conditions according to the Student's t-test ( p

    Article Snippet: Real-time quantitative PCR (qPCR) amplifications were performed in the LightCycler 480 (Roche) in a final volume of 6 µl containing 5 mM MgCl2 , 0.5 µM of each primer, 3 µl of reaction mix (LightCycler 480 SYBR Green I Master 2X) and 1 µl of each reverse transcribed RNA (diluted 1∶19).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Validation of RNA-Seq data using qRT-PCR. Correlation of expression levels of 12 selected DEGs derived from log 2 [fold change] in G_GG, G_GB, B_BB, and B_BG pairwise comparisons were determined by linear fitting the RNA-Seq and qRT-PCR data. Error bars represent the SD ( n = 3).

    Journal: International Journal of Molecular Sciences

    Article Title: Transcriptome Analysis Provides Insight into the Molecular Mechanisms Underlying gametophyte factor 2-Mediated Cross-Incompatibility in Maize

    doi: 10.3390/ijms19061757

    Figure Lengend Snippet: Validation of RNA-Seq data using qRT-PCR. Correlation of expression levels of 12 selected DEGs derived from log 2 [fold change] in G_GG, G_GB, B_BB, and B_BG pairwise comparisons were determined by linear fitting the RNA-Seq and qRT-PCR data. Error bars represent the SD ( n = 3).

    Article Snippet: The qRT-PCR amplification was performed on a LightCycler 480 II (Roche Life Science), using the following program: 95 °C for 10 min; followed by 40 cycles of 95 °C for 15 s, 60 °C for 10 s and 72 °C for 10 s; and a final extension step of 72 °C for 10 min.

    Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Derivative Assay