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<t>DNA</t> methylation of provirus is not associated with methylated CpG sites in the genome. Integration sites of HTLV-I provirus in leukemic cells have been determined by inverse <t>PCR,</t> and then DNA methylation in genome has been analyzed by sodium bisulfite sequencing. DNA methylation of 5'-LTR was also analyzed by sodium bisulfite sequencing method. Vertical bars represent CpG sites. Open circle indicates unmethylated CpG site, and closed one means methylated CpG site. N: normal PBMCs from non-carrier donor.
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1) Product Images from "Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms"

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms

Journal: Retrovirology

doi: 10.1186/1742-4690-2-64

DNA methylation of provirus is not associated with methylated CpG sites in the genome. Integration sites of HTLV-I provirus in leukemic cells have been determined by inverse PCR, and then DNA methylation in genome has been analyzed by sodium bisulfite sequencing. DNA methylation of 5'-LTR was also analyzed by sodium bisulfite sequencing method. Vertical bars represent CpG sites. Open circle indicates unmethylated CpG site, and closed one means methylated CpG site. N: normal PBMCs from non-carrier donor.
Figure Legend Snippet: DNA methylation of provirus is not associated with methylated CpG sites in the genome. Integration sites of HTLV-I provirus in leukemic cells have been determined by inverse PCR, and then DNA methylation in genome has been analyzed by sodium bisulfite sequencing. DNA methylation of 5'-LTR was also analyzed by sodium bisulfite sequencing method. Vertical bars represent CpG sites. Open circle indicates unmethylated CpG site, and closed one means methylated CpG site. N: normal PBMCs from non-carrier donor.

Techniques Used: DNA Methylation Assay, Methylation, Inverse PCR, Methylation Sequencing

DNA methylation and histone modifications in fresh ATL cases. A. The relationships among DNA methylation, tax gene expression and histone modification in 5'-LTR were analyzed in three ATL cases. Cases 1 and 3 have one copy of the complete HTLV-I provirus, while Case 2 has a defective provirus that lacks part of the pol gene. DNA methylation was analyzed by COBRA. The tax gene transcripts could be detected in Case 1, but not in Cases 2 or 3, by RT-PCR. ChIP assays were also performed using primers for 5'-LTR to analyze acetylation of histone H3 (Ac-H3) and H4 (Ac-H4). W.C.E.: whole cell extract. B. Recovery of tax gene expression ex vivo . The PBMCs isolated from Case 3 were immediately cultured ex vivo for several hours and tested the transcription of tax mRNA by RT-PCR.
Figure Legend Snippet: DNA methylation and histone modifications in fresh ATL cases. A. The relationships among DNA methylation, tax gene expression and histone modification in 5'-LTR were analyzed in three ATL cases. Cases 1 and 3 have one copy of the complete HTLV-I provirus, while Case 2 has a defective provirus that lacks part of the pol gene. DNA methylation was analyzed by COBRA. The tax gene transcripts could be detected in Case 1, but not in Cases 2 or 3, by RT-PCR. ChIP assays were also performed using primers for 5'-LTR to analyze acetylation of histone H3 (Ac-H3) and H4 (Ac-H4). W.C.E.: whole cell extract. B. Recovery of tax gene expression ex vivo . The PBMCs isolated from Case 3 were immediately cultured ex vivo for several hours and tested the transcription of tax mRNA by RT-PCR.

Techniques Used: DNA Methylation Assay, Expressing, Modification, Combined Bisulfite Restriction Analysis Assay, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Ex Vivo, Isolation, Cell Culture

DNA methylation in ATL cell lines, HTLV-I carriers and ATL cases. The tax gene transcription in ATL cell lines was studied by RT-PCR (A), and the expression of GAPDH gene has been used as a control. DNA methylation throughout the HTLV-I provirus was studied by COBRA in tax gene-expressing (B) and non-expressing cell lines (C). Furthermore, DNA methylation was also analyzed in 20 carriers and 20 ATL cases by COBRA, and representative patterns of DNA methylation are shown in D. The number of HTLV-I provirus has been analyzed by Southern blot method, and shown in the parenthesis (B, C and D). Each bar indicates the extent of DNA methylation that was calculated by COBRA.
Figure Legend Snippet: DNA methylation in ATL cell lines, HTLV-I carriers and ATL cases. The tax gene transcription in ATL cell lines was studied by RT-PCR (A), and the expression of GAPDH gene has been used as a control. DNA methylation throughout the HTLV-I provirus was studied by COBRA in tax gene-expressing (B) and non-expressing cell lines (C). Furthermore, DNA methylation was also analyzed in 20 carriers and 20 ATL cases by COBRA, and representative patterns of DNA methylation are shown in D. The number of HTLV-I provirus has been analyzed by Southern blot method, and shown in the parenthesis (B, C and D). Each bar indicates the extent of DNA methylation that was calculated by COBRA.

Techniques Used: DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Combined Bisulfite Restriction Analysis Assay, Southern Blot

Comparison of the DNA methylation in carriers and ATL cases. A. DNA methylation at eight different regions in the HTLV-I provirus was compared between carriers (C) and ATL cases (A). DNA methylation was quantified by COBRA in 20 carriers and 20 ATL cases. Each sample was analyzed three times by COBRA at each site, and circles indicate mean values of DNA methylation. The differences of DNA methylation are statistically significant in the gag , pol and env regions by the Mann-Whitney's U-test. Horizontal bars represent median of DNA methylation in each group. B. The relation between tax gene transcription and DNA methylation of 5'-LTR in the fresh ATL cells has been shown. DNA methylation of 5'-LTR was quantified by COBRA assay and the tax gene transcripts were detected by RT-PCR.
Figure Legend Snippet: Comparison of the DNA methylation in carriers and ATL cases. A. DNA methylation at eight different regions in the HTLV-I provirus was compared between carriers (C) and ATL cases (A). DNA methylation was quantified by COBRA in 20 carriers and 20 ATL cases. Each sample was analyzed three times by COBRA at each site, and circles indicate mean values of DNA methylation. The differences of DNA methylation are statistically significant in the gag , pol and env regions by the Mann-Whitney's U-test. Horizontal bars represent median of DNA methylation in each group. B. The relation between tax gene transcription and DNA methylation of 5'-LTR in the fresh ATL cells has been shown. DNA methylation of 5'-LTR was quantified by COBRA assay and the tax gene transcripts were detected by RT-PCR.

Techniques Used: DNA Methylation Assay, Combined Bisulfite Restriction Analysis Assay, MANN-WHITNEY, Reverse Transcription Polymerase Chain Reaction

DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.
Figure Legend Snippet: DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.

Techniques Used: DNA Methylation Assay, Methylation Sequencing, Combined Bisulfite Restriction Analysis Assay, Expressing, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Methylation

2) Product Images from "Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms"

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms

Journal: Retrovirology

doi: 10.1186/1742-4690-2-64

DNA methylation of provirus is not associated with methylated CpG sites in the genome. Integration sites of HTLV-I provirus in leukemic cells have been determined by inverse PCR, and then DNA methylation in genome has been analyzed by sodium bisulfite sequencing. DNA methylation of 5'-LTR was also analyzed by sodium bisulfite sequencing method. Vertical bars represent CpG sites. Open circle indicates unmethylated CpG site, and closed one means methylated CpG site. N: normal PBMCs from non-carrier donor.
Figure Legend Snippet: DNA methylation of provirus is not associated with methylated CpG sites in the genome. Integration sites of HTLV-I provirus in leukemic cells have been determined by inverse PCR, and then DNA methylation in genome has been analyzed by sodium bisulfite sequencing. DNA methylation of 5'-LTR was also analyzed by sodium bisulfite sequencing method. Vertical bars represent CpG sites. Open circle indicates unmethylated CpG site, and closed one means methylated CpG site. N: normal PBMCs from non-carrier donor.

Techniques Used: DNA Methylation Assay, Methylation, Inverse PCR, Methylation Sequencing

DNA methylation and histone modifications in fresh ATL cases. A. The relationships among DNA methylation, tax gene expression and histone modification in 5'-LTR were analyzed in three ATL cases. Cases 1 and 3 have one copy of the complete HTLV-I provirus, while Case 2 has a defective provirus that lacks part of the pol gene. DNA methylation was analyzed by COBRA. The tax gene transcripts could be detected in Case 1, but not in Cases 2 or 3, by RT-PCR. ChIP assays were also performed using primers for 5'-LTR to analyze acetylation of histone H3 (Ac-H3) and H4 (Ac-H4). W.C.E.: whole cell extract. B. Recovery of tax gene expression ex vivo . The PBMCs isolated from Case 3 were immediately cultured ex vivo for several hours and tested the transcription of tax mRNA by RT-PCR.
Figure Legend Snippet: DNA methylation and histone modifications in fresh ATL cases. A. The relationships among DNA methylation, tax gene expression and histone modification in 5'-LTR were analyzed in three ATL cases. Cases 1 and 3 have one copy of the complete HTLV-I provirus, while Case 2 has a defective provirus that lacks part of the pol gene. DNA methylation was analyzed by COBRA. The tax gene transcripts could be detected in Case 1, but not in Cases 2 or 3, by RT-PCR. ChIP assays were also performed using primers for 5'-LTR to analyze acetylation of histone H3 (Ac-H3) and H4 (Ac-H4). W.C.E.: whole cell extract. B. Recovery of tax gene expression ex vivo . The PBMCs isolated from Case 3 were immediately cultured ex vivo for several hours and tested the transcription of tax mRNA by RT-PCR.

Techniques Used: DNA Methylation Assay, Expressing, Modification, Combined Bisulfite Restriction Analysis Assay, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Ex Vivo, Isolation, Cell Culture

DNA methylation in ATL cell lines, HTLV-I carriers and ATL cases. The tax gene transcription in ATL cell lines was studied by RT-PCR (A), and the expression of GAPDH gene has been used as a control. DNA methylation throughout the HTLV-I provirus was studied by COBRA in tax gene-expressing (B) and non-expressing cell lines (C). Furthermore, DNA methylation was also analyzed in 20 carriers and 20 ATL cases by COBRA, and representative patterns of DNA methylation are shown in D. The number of HTLV-I provirus has been analyzed by Southern blot method, and shown in the parenthesis (B, C and D). Each bar indicates the extent of DNA methylation that was calculated by COBRA.
Figure Legend Snippet: DNA methylation in ATL cell lines, HTLV-I carriers and ATL cases. The tax gene transcription in ATL cell lines was studied by RT-PCR (A), and the expression of GAPDH gene has been used as a control. DNA methylation throughout the HTLV-I provirus was studied by COBRA in tax gene-expressing (B) and non-expressing cell lines (C). Furthermore, DNA methylation was also analyzed in 20 carriers and 20 ATL cases by COBRA, and representative patterns of DNA methylation are shown in D. The number of HTLV-I provirus has been analyzed by Southern blot method, and shown in the parenthesis (B, C and D). Each bar indicates the extent of DNA methylation that was calculated by COBRA.

Techniques Used: DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Combined Bisulfite Restriction Analysis Assay, Southern Blot

Comparison of the DNA methylation in carriers and ATL cases. A. DNA methylation at eight different regions in the HTLV-I provirus was compared between carriers (C) and ATL cases (A). DNA methylation was quantified by COBRA in 20 carriers and 20 ATL cases. Each sample was analyzed three times by COBRA at each site, and circles indicate mean values of DNA methylation. The differences of DNA methylation are statistically significant in the gag , pol and env regions by the Mann-Whitney's U-test. Horizontal bars represent median of DNA methylation in each group. B. The relation between tax gene transcription and DNA methylation of 5'-LTR in the fresh ATL cells has been shown. DNA methylation of 5'-LTR was quantified by COBRA assay and the tax gene transcripts were detected by RT-PCR.
Figure Legend Snippet: Comparison of the DNA methylation in carriers and ATL cases. A. DNA methylation at eight different regions in the HTLV-I provirus was compared between carriers (C) and ATL cases (A). DNA methylation was quantified by COBRA in 20 carriers and 20 ATL cases. Each sample was analyzed three times by COBRA at each site, and circles indicate mean values of DNA methylation. The differences of DNA methylation are statistically significant in the gag , pol and env regions by the Mann-Whitney's U-test. Horizontal bars represent median of DNA methylation in each group. B. The relation between tax gene transcription and DNA methylation of 5'-LTR in the fresh ATL cells has been shown. DNA methylation of 5'-LTR was quantified by COBRA assay and the tax gene transcripts were detected by RT-PCR.

Techniques Used: DNA Methylation Assay, Combined Bisulfite Restriction Analysis Assay, MANN-WHITNEY, Reverse Transcription Polymerase Chain Reaction

DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.
Figure Legend Snippet: DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.

Techniques Used: DNA Methylation Assay, Methylation Sequencing, Combined Bisulfite Restriction Analysis Assay, Expressing, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Methylation

3) Product Images from "Lanthanide-dependent cross-feeding of methane-derived carbon is linked by microbial community interactions"

Article Title: Lanthanide-dependent cross-feeding of methane-derived carbon is linked by microbial community interactions

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1619871114

RNA Extraction and qRT-PCR.
Figure Legend Snippet: RNA Extraction and qRT-PCR.

Techniques Used: RNA Extraction, Quantitative RT-PCR

qRT-PCR values depicting the fold change in the expression of M . tundripaludum 31/32 mxaF and xoxF genes. ( A ) In pure culture with different concentrations of lanthanum chloride or without lanthanum ( n = 2 for each concentration). ( B ) In pure culture
Figure Legend Snippet: qRT-PCR values depicting the fold change in the expression of M . tundripaludum 31/32 mxaF and xoxF genes. ( A ) In pure culture with different concentrations of lanthanum chloride or without lanthanum ( n = 2 for each concentration). ( B ) In pure culture

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

qRT-PCR values depicting the fold change in the expression of M . tundripaludum 31/32 mxaF (black bars) and xoxF (gray bars) genes in pure culture with lanthanum chloride (30 μM) and 10 mL supernatant from cocultures of M . tundripaludum 31/32 and
Figure Legend Snippet: qRT-PCR values depicting the fold change in the expression of M . tundripaludum 31/32 mxaF (black bars) and xoxF (gray bars) genes in pure culture with lanthanum chloride (30 μM) and 10 mL supernatant from cocultures of M . tundripaludum 31/32 and

Techniques Used: Quantitative RT-PCR, Expressing

( A ) Cell-abundance dynamics of individual species in model artificial communities. ( B ) qRT-PCR values depicting the fold change in mxaF and xoxF gene expression from M . tundripaludum 31/32 with these cocultures. Cocultures were grown on methane as the
Figure Legend Snippet: ( A ) Cell-abundance dynamics of individual species in model artificial communities. ( B ) qRT-PCR values depicting the fold change in mxaF and xoxF gene expression from M . tundripaludum 31/32 with these cocultures. Cocultures were grown on methane as the

Techniques Used: Quantitative RT-PCR, Expressing

qRT-PCR values depicting the fold change in expression of M . tundripaludum 31/32 mxaF and xoxF genes in pure culture with or without 30 μM lanthanum chloride ( A ), in the presence or absence of M . mobilis 13 (both with 30 μM lanthanum chloride)
Figure Legend Snippet: qRT-PCR values depicting the fold change in expression of M . tundripaludum 31/32 mxaF and xoxF genes in pure culture with or without 30 μM lanthanum chloride ( A ), in the presence or absence of M . mobilis 13 (both with 30 μM lanthanum chloride)

Techniques Used: Quantitative RT-PCR, Expressing

4) Product Images from "Synergism between DNA methylation and macroH2A1 occupancy in epigenetic silencing of the tumor suppressor gene p16(CDKN2A)"

Article Title: Synergism between DNA methylation and macroH2A1 occupancy in epigenetic silencing of the tumor suppressor gene p16(CDKN2A)

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq994

Silencing of CDKN2A is accompanied by macroH2A1 enrichment. ( A ) Expression and methylation status of CDKN2A in early passage (p18) and late passage (p32) WI-38 cell: western blot for p16 and β-actin showing loss of expression in p32 cells (left panel). Methylation of the CDKN2A promoter in p32 cell confirmed by methylation-specific PCR (MSP) on bisulfite-modified DNA (right panel). ( B ) ChIP with macroH2A1 antibody. The bound/input ratio for each cell line was calculated using quantitative real-time PCR. To compare between cell lines, enrichment of the bound fraction was normalized according to the positive control α-Crystallin, which is inactive in both cultures ( Supplementary Figure S6 ). An 8.5-fold enrichment for macroH2A1 at the CDKN2A promoter is observed in p32 cells versus p18 cells. Similar results were observed in three biological replicates (see also Figure 5 ). HoxA9 is another positive control and is not expressed. Aprt and MLH1 are expressed in both samples. The numbers under the gene names indicate position of the interrogated region relative to TSS. Error bars represent standard deviation. ( C ) ChIP analysis using anti-macroH2A1 or anti-acetylated H3 antibody on HCT116 cells. Input and bound fractions were analyzed by SNaPshot assay to discriminate between the silenced (wild-type) allele of CDKN2A (S) and the active mutant allele (A). The ratio between the two alleles (S/A ratio) was determined based on peak area using Genotyper 2.1 software (ABI PRISM).
Figure Legend Snippet: Silencing of CDKN2A is accompanied by macroH2A1 enrichment. ( A ) Expression and methylation status of CDKN2A in early passage (p18) and late passage (p32) WI-38 cell: western blot for p16 and β-actin showing loss of expression in p32 cells (left panel). Methylation of the CDKN2A promoter in p32 cell confirmed by methylation-specific PCR (MSP) on bisulfite-modified DNA (right panel). ( B ) ChIP with macroH2A1 antibody. The bound/input ratio for each cell line was calculated using quantitative real-time PCR. To compare between cell lines, enrichment of the bound fraction was normalized according to the positive control α-Crystallin, which is inactive in both cultures ( Supplementary Figure S6 ). An 8.5-fold enrichment for macroH2A1 at the CDKN2A promoter is observed in p32 cells versus p18 cells. Similar results were observed in three biological replicates (see also Figure 5 ). HoxA9 is another positive control and is not expressed. Aprt and MLH1 are expressed in both samples. The numbers under the gene names indicate position of the interrogated region relative to TSS. Error bars represent standard deviation. ( C ) ChIP analysis using anti-macroH2A1 or anti-acetylated H3 antibody on HCT116 cells. Input and bound fractions were analyzed by SNaPshot assay to discriminate between the silenced (wild-type) allele of CDKN2A (S) and the active mutant allele (A). The ratio between the two alleles (S/A ratio) was determined based on peak area using Genotyper 2.1 software (ABI PRISM).

Techniques Used: Expressing, Methylation, Western Blot, Polymerase Chain Reaction, Modification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Standard Deviation, Mutagenesis, Software

5) Product Images from "IB4-binding sensory neurons in the adult rat express a novel 3′ UTR-extended isoform of CaMK4 that is associated with its localization to axons"

Article Title: IB4-binding sensory neurons in the adult rat express a novel 3′ UTR-extended isoform of CaMK4 that is associated with its localization to axons

Journal: The Journal of comparative neurology

doi: 10.1002/cne.23398

Characterization of a novel 3'UTR extended CaMK4 mRNA transcript expressed in rat cutaneous nociceptors A: Summary table of alternate polyadenylation signal characterisation experiments: The POLYAR algorithm was used to predict the position of polyadenylation signals on the final exon of CaMK4 +12kb downstream. Integral (W) scores, cleavage site (CS) and polyadenylation specificity signal (PAS) positions are relative to the start codon. Rapid amplification of cDNA ends (RACE) was performed using 7 distinct primers designed 200–900bp upstream of the predicated cleavage site. B: PCR products were separated by electrophoresis and any observable bands excised for sequencing. Bands with sequences that mapped to CaMK4 were validated polyadenylation sites (arrows). C: To determine if the extended UTR region was contiguous with the coding sequence (CDS), we designed a gene specific primer (GSP) to prime the cDNA synthesis reaction. PCR probing the kinase domain of CaMK4 demonstrated that the extended UTR was part of the same transcript as the CDS. cDNA synthesised without primer (-P) was employed as a negative control. D: UCSC genome browser track centered on the validated polyadenylation site discovered using primer RACE_7. A cluster of expressed sequence tags (ESTs - black lines) terminate at this highly conserved CS which is ≈ 10kb downstream of the CaMKIV stop codon. The CS is flanked by conserved putative PAS and U-/GU-rich down-stream element (DSE) sequence motifs.
Figure Legend Snippet: Characterization of a novel 3'UTR extended CaMK4 mRNA transcript expressed in rat cutaneous nociceptors A: Summary table of alternate polyadenylation signal characterisation experiments: The POLYAR algorithm was used to predict the position of polyadenylation signals on the final exon of CaMK4 +12kb downstream. Integral (W) scores, cleavage site (CS) and polyadenylation specificity signal (PAS) positions are relative to the start codon. Rapid amplification of cDNA ends (RACE) was performed using 7 distinct primers designed 200–900bp upstream of the predicated cleavage site. B: PCR products were separated by electrophoresis and any observable bands excised for sequencing. Bands with sequences that mapped to CaMK4 were validated polyadenylation sites (arrows). C: To determine if the extended UTR region was contiguous with the coding sequence (CDS), we designed a gene specific primer (GSP) to prime the cDNA synthesis reaction. PCR probing the kinase domain of CaMK4 demonstrated that the extended UTR was part of the same transcript as the CDS. cDNA synthesised without primer (-P) was employed as a negative control. D: UCSC genome browser track centered on the validated polyadenylation site discovered using primer RACE_7. A cluster of expressed sequence tags (ESTs - black lines) terminate at this highly conserved CS which is ≈ 10kb downstream of the CaMKIV stop codon. The CS is flanked by conserved putative PAS and U-/GU-rich down-stream element (DSE) sequence motifs.

Techniques Used: Rapid Amplification of cDNA Ends, Polymerase Chain Reaction, Electrophoresis, Sequencing, Negative Control

6) Product Images from "Adjusting microbiome profiles for differences in microbial load by spike-in bacteria"

Article Title: Adjusting microbiome profiles for differences in microbial load by spike-in bacteria

Journal: Microbiome

doi: 10.1186/s40168-016-0175-0

Procedural overview of proposed spike-in procedure and the spike-in-based calibration to total microbial load (SCML). The overview is divided into four sections: spike-in procedure and bacterial lysis (blue), DNA isolation, amplification and sequencing (yellow), pre-processing (red) and the actual spike-in-based calibration to microbial load (green). White-filled boxes depict procedural intermediates, while grey-filled boxes depict the different procedural steps. Each step is numbered. In the first step (1) whole cells of exogenous spike bacteria corresponding to a fixed number of 16S rDNA copies are added to homogenized microbiome samples. Bacterial lysis is performed on the resulting spiked samples (2). Metagenomic DNA is extracted from the lysates (3) and PCR amplified using 16S rDNA specific primers (4), creating 16S rDNA amplicons. These amplicons are purified and pyrosequencing is performed (5). The resulting raw read counts are pre-processed with QIIME (quality filtering, demultiplexing and closed reference OTU picking) to generate OTU read count tables (6). Based on the read counts associated with single or multiple reference spike-in bacteria, a size factor s i for each sample i is calculated and applied to each OTU of this particular sample i (8, see methods section). This leads to an OTU read count table calibrated to differences in microbial load. These read counts can be utilized to more accurately assess changes between different samples. All depicted steps are described in detail in the methods section. Stars indicate points in the procedure at which qPCR is performed to identify possible errors in DNA isolation (metagenomic DNA) or PCR amplification (16S rDNA amplicons).
Figure Legend Snippet: Procedural overview of proposed spike-in procedure and the spike-in-based calibration to total microbial load (SCML). The overview is divided into four sections: spike-in procedure and bacterial lysis (blue), DNA isolation, amplification and sequencing (yellow), pre-processing (red) and the actual spike-in-based calibration to microbial load (green). White-filled boxes depict procedural intermediates, while grey-filled boxes depict the different procedural steps. Each step is numbered. In the first step (1) whole cells of exogenous spike bacteria corresponding to a fixed number of 16S rDNA copies are added to homogenized microbiome samples. Bacterial lysis is performed on the resulting spiked samples (2). Metagenomic DNA is extracted from the lysates (3) and PCR amplified using 16S rDNA specific primers (4), creating 16S rDNA amplicons. These amplicons are purified and pyrosequencing is performed (5). The resulting raw read counts are pre-processed with QIIME (quality filtering, demultiplexing and closed reference OTU picking) to generate OTU read count tables (6). Based on the read counts associated with single or multiple reference spike-in bacteria, a size factor s i for each sample i is calculated and applied to each OTU of this particular sample i (8, see methods section). This leads to an OTU read count table calibrated to differences in microbial load. These read counts can be utilized to more accurately assess changes between different samples. All depicted steps are described in detail in the methods section. Stars indicate points in the procedure at which qPCR is performed to identify possible errors in DNA isolation (metagenomic DNA) or PCR amplification (16S rDNA amplicons).

Techniques Used: Lysis, DNA Extraction, Amplification, Sequencing, Polymerase Chain Reaction, Purification, Real-time Polymerase Chain Reaction

Comparison of SCML and normalization by qRT-PCR-derived total number of 16S rDNA copies to all background OTUs. Observed log 2 ratio versus expected log 2 ratio of all background bacteria OTUs for all pairwise sample comparisons after ( a ) SCML by S. ruber and ( b ) normalization by qRT-PCR derived total 16S rDNA copy number. The data is binned to hexagons because of the high number of data points. The colour of each hexagon represents the percentage of all counts at the corresponding level of expected log 2 ratios contained in each bin. Bins that contributed to less than 0.05 percent for each level of expected log 2 ratio are omitted. The purple diagonal represents the identity, which represents the expected log 2 ratios by design. The box-plots in ( c ) summarize the error between the expected and observed log 2 ratios for the four different approaches. The smaller this error, the better calibrated the ratios are. Variances of the log 2 differences are 3.65, 2.01, 1.28 and 1.18 as derived from relative abundances, counts calibrated for differences in total number of 16S rRNA gene copies, SCML (by S. ruber ) and combined SCML (by S. ruber , A. acidiphilus and R. radiobacter ), respectively
Figure Legend Snippet: Comparison of SCML and normalization by qRT-PCR-derived total number of 16S rDNA copies to all background OTUs. Observed log 2 ratio versus expected log 2 ratio of all background bacteria OTUs for all pairwise sample comparisons after ( a ) SCML by S. ruber and ( b ) normalization by qRT-PCR derived total 16S rDNA copy number. The data is binned to hexagons because of the high number of data points. The colour of each hexagon represents the percentage of all counts at the corresponding level of expected log 2 ratios contained in each bin. Bins that contributed to less than 0.05 percent for each level of expected log 2 ratio are omitted. The purple diagonal represents the identity, which represents the expected log 2 ratios by design. The box-plots in ( c ) summarize the error between the expected and observed log 2 ratios for the four different approaches. The smaller this error, the better calibrated the ratios are. Variances of the log 2 differences are 3.65, 2.01, 1.28 and 1.18 as derived from relative abundances, counts calibrated for differences in total number of 16S rRNA gene copies, SCML (by S. ruber ) and combined SCML (by S. ruber , A. acidiphilus and R. radiobacter ), respectively

Techniques Used: Quantitative RT-PCR, Derivative Assay

7) Product Images from "Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering"

Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

Journal: Scientific Reports

doi: 10.1038/s41598-018-33242-z

Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.
Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.

Techniques Used: Expressing, Quantitative RT-PCR

Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.
Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.

Techniques Used: Expressing, Quantitative RT-PCR

Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.
Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.

Techniques Used: Expressing, Quantitative RT-PCR

Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.
Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.

Techniques Used: Expressing, Quantitative RT-PCR

8) Product Images from "A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis"

Article Title: A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis

Journal: BMC Microbiology

doi:

PCR amplification of B. anthracis minisatellite CEB-Bams30 DNA from B. anthracis and B. cereus (six rightmost lanes) was amplified using primers for CEB-Bams30 (Table 2 ). The PCR products were run on a 40 cm long 2% ordinary agarose gel.
Figure Legend Snippet: PCR amplification of B. anthracis minisatellite CEB-Bams30 DNA from B. anthracis and B. cereus (six rightmost lanes) was amplified using primers for CEB-Bams30 (Table 2 ). The PCR products were run on a 40 cm long 2% ordinary agarose gel.

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

Selection procedure of minisatellites for Y. pestis 3A: Sixty-four tandem repeats have at least 7 units longer than 9 base-pairs. Panel A presents the distribution of these 64 loci according to repeat unit length. Each rectangle is an hyperlink to an alignment file. The rectangle indicated by the arrow is linked to the file illustrated in panel B. 3B: This is an annotated alignment file. The file corresponds to Yp3057ms09 (Table 1 and Figure 4 ; Yp : Yersinia pestis ; 3057 : position on the genome, expressed in kilobases; MS09 : MiniSatellite index). The consensus pattern of 18 base-pairs is aligned to each motif. Annotations of the file are inserted within brackets. Although this minisatellite is very polymorphic, eleven different motifs (labeled a-k) are observed in the sequenced allele. The first four and last two copies are most diverged and rare. Four types of motifs (f, g, h, i) constitute most of the array. For convenience, 18 motifs have been removed from the alignment file and replaced by their letter code. The last two copies are 21 base-pair long instead of 18. The end of the alignment file (panel B, bottom) provides sequence data flanking the tandem repeat array. The positions of the primers chosen for PCR amplification of this locus (Table 1 ) are shown underlined.
Figure Legend Snippet: Selection procedure of minisatellites for Y. pestis 3A: Sixty-four tandem repeats have at least 7 units longer than 9 base-pairs. Panel A presents the distribution of these 64 loci according to repeat unit length. Each rectangle is an hyperlink to an alignment file. The rectangle indicated by the arrow is linked to the file illustrated in panel B. 3B: This is an annotated alignment file. The file corresponds to Yp3057ms09 (Table 1 and Figure 4 ; Yp : Yersinia pestis ; 3057 : position on the genome, expressed in kilobases; MS09 : MiniSatellite index). The consensus pattern of 18 base-pairs is aligned to each motif. Annotations of the file are inserted within brackets. Although this minisatellite is very polymorphic, eleven different motifs (labeled a-k) are observed in the sequenced allele. The first four and last two copies are most diverged and rare. Four types of motifs (f, g, h, i) constitute most of the array. For convenience, 18 motifs have been removed from the alignment file and replaced by their letter code. The last two copies are 21 base-pair long instead of 18. The end of the alignment file (panel B, bottom) provides sequence data flanking the tandem repeat array. The positions of the primers chosen for PCR amplification of this locus (Table 1 ) are shown underlined.

Techniques Used: Selection, Labeling, Sequencing, Polymerase Chain Reaction, Amplification

Images of PCR amplification of the twenty-five minisatellites polymorphic in the Y. pestis strains DNA from three reference Y. pestis strains representing each of the main biovars, antiqua (lane 1), medievalis (lane 2) and orientalis (lane 3) and two Y. pseudotuberculosis strains (lanes 4 and 5) have been PCR amplified and an aliquot of the products has been run on 2% horizontal agarose gels as described. The length of the minisatellite motifs (U) and the size range is indicated on each panel. Yp2916ms07 has one of the shortest (10 bp) unit. Four alleles are clearly distinguished between the 150 and 200 bp marker fragments.
Figure Legend Snippet: Images of PCR amplification of the twenty-five minisatellites polymorphic in the Y. pestis strains DNA from three reference Y. pestis strains representing each of the main biovars, antiqua (lane 1), medievalis (lane 2) and orientalis (lane 3) and two Y. pseudotuberculosis strains (lanes 4 and 5) have been PCR amplified and an aliquot of the products has been run on 2% horizontal agarose gels as described. The length of the minisatellite motifs (U) and the size range is indicated on each panel. Yp2916ms07 has one of the shortest (10 bp) unit. Four alleles are clearly distinguished between the 150 and 200 bp marker fragments.

Techniques Used: Polymerase Chain Reaction, Amplification, Marker

Bacillus anthracis phylogenetic tree The genotype of each strain for the polymorphic minisatellites is given (size estimates for each allele are given in Table 3 ).
Figure Legend Snippet: Bacillus anthracis phylogenetic tree The genotype of each strain for the polymorphic minisatellites is given (size estimates for each allele are given in Table 3 ). "0" indicates a failure of the PCR amplification. This is most often associated with B. cereus strains, and probably reflects in these cases sequence divergence in the flanking sequence. The phylogenetic tree was produced using the Neighbor-Joining method as available on-line at

Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Produced

9) Product Images from "Contribution of ARLTS1 Cys148Arg (T442C) Variant with Prostate Cancer Risk and ARLTS1 Function in Prostate Cancer Cells"

Article Title: Contribution of ARLTS1 Cys148Arg (T442C) Variant with Prostate Cancer Risk and ARLTS1 Function in Prostate Cancer Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0026595

Relative expression of ARLTS1 in A, LuCaP xenografts, prostate cancer cell lines, prostate epithelial cells and in normal prostate and B, in BPH and clinical tumor samples, as determined by Q-RT-PCR. The Cys148Arg (T442C) genotype as well as the choromosomal aberration is shown. C , Cys148Arg (T442C) variant in two clinical cancer tissue samples and in two normal blood DNA (sequences are in reverse orientation). * determined as in Saramäki OR et al., 2006.
Figure Legend Snippet: Relative expression of ARLTS1 in A, LuCaP xenografts, prostate cancer cell lines, prostate epithelial cells and in normal prostate and B, in BPH and clinical tumor samples, as determined by Q-RT-PCR. The Cys148Arg (T442C) genotype as well as the choromosomal aberration is shown. C , Cys148Arg (T442C) variant in two clinical cancer tissue samples and in two normal blood DNA (sequences are in reverse orientation). * determined as in Saramäki OR et al., 2006.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Variant Assay

10) Product Images from "Coordinated transcriptional regulation of bone homeostasis by Ebf1 and Zfp521 in both mesenchymal and hematopoietic lineages"

Article Title: Coordinated transcriptional regulation of bone homeostasis by Ebf1 and Zfp521 in both mesenchymal and hematopoietic lineages

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20121187

Zfp521 favors bone formation in mature OBs. (A) Generation of null and conditional Zfp521 alleles. Zfp521 genomic region encoding exon 4 is shown. Restriction fragment sizes are indicated as well as the positions of the internal and the two flanking probes used for genotyping analysis (Roman numerals). The shaded area indicates the part of the genomic region included in the targeting vector, and the three different alleles are shown. “neo” is the result of the gene-targeting event. “cko” is the conditional knockout allele derived from the neo allele after Flpe-mediated excision of the PGK-neo cassette. One Frt site and two loxP sites remain in the locus. “ko” is the null allele derived from the “neo” or from the “cko” allele by Cre-mediated recombination between the two loxP sites. Only a single loxP site remains in the modified locus. Splicing of exon 3 to exon 5 generates a frameshift. loxP and Frt sites are indicated as closed and open triangles, respectively. Neo, PGK-em7-neomycin dual selection cassette for bacteria and embryonic stem cells; TK, thymidine kinase cassette for counter-selection in embryonic stem cells; X, XbaI; B, BamHI; N, NotI. The genomic region is not drawn to scale. (B) Results of a Southern blot analysis of BamHI-digested tail DNA, probed with the internal probe (III). (C) Northern blot analysis of whole-brain RNA from 3-wk-old mice using a full-length Zfp521 cDNA probe. The blot was rehybridized with a Gapdh probe as a control for RNA quality. wt, wild type; Δexon4, position of the residual mRNA after removal of exon 4. (D) Genotyping PCR showing deletion of Zfp521 allele in genomic DNA extracted from Zfp521 hOC −/− long bones cleaned of soft tissues and BM. (E) Von Kossa staining of tibia sections in 3-wk-old global Zfp521 −/− mice and Zfp521 +/+ littermate controls. (F) Histomorphometric analysis of samples in E ( n = 5). (G) Trabecular BV (BV/tissue volume [TV]) at distal femoral metaphysis and in second lumbar vertebra in 3-wk-old Zfp521 −/− and control mice measured by μCT ( n = 5). (H) Von Kossa staining of tibia sections in 6-wk-old Zfp521 hOC −/− mice and littermate controls. (I) Histomorphometric analysis of samples in H ( n = 6). (J) Trabecular BV (BV/TV) at distal femoral metaphysis and in second lumbar vertebra in 12-wk-old Zfp521 hOC −/− and control mice measured by μCT ( n = 5–6). (K) Von Kossa staining of tibia sections in 12-wk-old Zfp521 hOC −/− mice and littermate controls. (L) Histomorphometric analysis of samples in K ( n = 6). (M) Serum PINP and CTX levels in 3-wk-old global Zfp521 −/− mice and Zfp521 +/+ littermate controls ( n = 6–9). (N) Serum PINP and CTX levels in 6-wk-old Zfp521 hOC −/− and control mice ( n = 5–6). N.Ob, number of OBs; N.Oc, number of OCs. All data are means ± SD. *, P
Figure Legend Snippet: Zfp521 favors bone formation in mature OBs. (A) Generation of null and conditional Zfp521 alleles. Zfp521 genomic region encoding exon 4 is shown. Restriction fragment sizes are indicated as well as the positions of the internal and the two flanking probes used for genotyping analysis (Roman numerals). The shaded area indicates the part of the genomic region included in the targeting vector, and the three different alleles are shown. “neo” is the result of the gene-targeting event. “cko” is the conditional knockout allele derived from the neo allele after Flpe-mediated excision of the PGK-neo cassette. One Frt site and two loxP sites remain in the locus. “ko” is the null allele derived from the “neo” or from the “cko” allele by Cre-mediated recombination between the two loxP sites. Only a single loxP site remains in the modified locus. Splicing of exon 3 to exon 5 generates a frameshift. loxP and Frt sites are indicated as closed and open triangles, respectively. Neo, PGK-em7-neomycin dual selection cassette for bacteria and embryonic stem cells; TK, thymidine kinase cassette for counter-selection in embryonic stem cells; X, XbaI; B, BamHI; N, NotI. The genomic region is not drawn to scale. (B) Results of a Southern blot analysis of BamHI-digested tail DNA, probed with the internal probe (III). (C) Northern blot analysis of whole-brain RNA from 3-wk-old mice using a full-length Zfp521 cDNA probe. The blot was rehybridized with a Gapdh probe as a control for RNA quality. wt, wild type; Δexon4, position of the residual mRNA after removal of exon 4. (D) Genotyping PCR showing deletion of Zfp521 allele in genomic DNA extracted from Zfp521 hOC −/− long bones cleaned of soft tissues and BM. (E) Von Kossa staining of tibia sections in 3-wk-old global Zfp521 −/− mice and Zfp521 +/+ littermate controls. (F) Histomorphometric analysis of samples in E ( n = 5). (G) Trabecular BV (BV/tissue volume [TV]) at distal femoral metaphysis and in second lumbar vertebra in 3-wk-old Zfp521 −/− and control mice measured by μCT ( n = 5). (H) Von Kossa staining of tibia sections in 6-wk-old Zfp521 hOC −/− mice and littermate controls. (I) Histomorphometric analysis of samples in H ( n = 6). (J) Trabecular BV (BV/TV) at distal femoral metaphysis and in second lumbar vertebra in 12-wk-old Zfp521 hOC −/− and control mice measured by μCT ( n = 5–6). (K) Von Kossa staining of tibia sections in 12-wk-old Zfp521 hOC −/− mice and littermate controls. (L) Histomorphometric analysis of samples in K ( n = 6). (M) Serum PINP and CTX levels in 3-wk-old global Zfp521 −/− mice and Zfp521 +/+ littermate controls ( n = 6–9). (N) Serum PINP and CTX levels in 6-wk-old Zfp521 hOC −/− and control mice ( n = 5–6). N.Ob, number of OBs; N.Oc, number of OCs. All data are means ± SD. *, P

Techniques Used: Plasmid Preparation, Knock-Out, Derivative Assay, Modification, Selection, Southern Blot, Northern Blot, Mouse Assay, Polymerase Chain Reaction, Staining

11) Product Images from "Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering"

Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

Journal: Scientific Reports

doi: 10.1038/s41598-018-33242-z

Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.
Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.

Techniques Used: Expressing, Quantitative RT-PCR

Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.
Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.

Techniques Used: Expressing, Quantitative RT-PCR

Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.
Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.

Techniques Used: Expressing, Quantitative RT-PCR

Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) untreated rat rectus abdominis muscle tissue, ( B ) rat rectus abdominis muscle tissue treated with osteogenic medium, and ( C ) both normal and treated rat muscle tissue.
Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) untreated rat rectus abdominis muscle tissue, ( B ) rat rectus abdominis muscle tissue treated with osteogenic medium, and ( C ) both normal and treated rat muscle tissue.

Techniques Used: Expressing, Quantitative RT-PCR

Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.
Figure Legend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.

Techniques Used: Expressing, Quantitative RT-PCR

12) Product Images from "Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine"

Article Title: Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq223

Preparation and validation of modified oligonucleotides. ( A ) Sequence and preparation of the 76-mers used in the assays. The synthesized DNA fragments contain three BstUI sites (5′-CGCG, underlined). Two rounds of PCR were performed to obtain C76, 5mC76 or 5hmC76 containing C, 5mC or 5hmC at CpG sites. X indicates normal C, or modified bases 5mC or 5hmC. The boxed sequences indicate the PCR primers. Oligonucleotides containing both 5mC and 5hmC were synthesized chemically. ( B ) Analysis of PCR products C76, 5mC76 and 5hmC76 by BstUI cleavage. PCR products were prepared and digested with the methylation-sensitive restriction enzyme BstUI, then separated and visualized by electrophoresis on 3% Nusieve GTG agarose gels. Lane 1 (C76), lane 2 (5mC76) and lane 3 (5hmC76) show clean single bands for each of the PCR products. After BstUI digestion, C76 (lane 4) was fully digested, whereas samples in lane 5 (5mC76) and lane 6 (5hmC76) resisted digestion. ( C ) Analysis of PCR products C76, 5mC76 and 5hmC76 by cleavage with different methylation-sensitive restriction enzymes. C76 (lanes 1, 4, 7 and 10), 5mC76 (lanes 2, 5, 8 and 11) and 5hmC76 (lanes 3, 6, 9 and 12) were left untreated (lanes 1–3) or were incubated with MluI (lanes 4–6), NruI (lanes 7–9) or HhaI (lanes 9–12).
Figure Legend Snippet: Preparation and validation of modified oligonucleotides. ( A ) Sequence and preparation of the 76-mers used in the assays. The synthesized DNA fragments contain three BstUI sites (5′-CGCG, underlined). Two rounds of PCR were performed to obtain C76, 5mC76 or 5hmC76 containing C, 5mC or 5hmC at CpG sites. X indicates normal C, or modified bases 5mC or 5hmC. The boxed sequences indicate the PCR primers. Oligonucleotides containing both 5mC and 5hmC were synthesized chemically. ( B ) Analysis of PCR products C76, 5mC76 and 5hmC76 by BstUI cleavage. PCR products were prepared and digested with the methylation-sensitive restriction enzyme BstUI, then separated and visualized by electrophoresis on 3% Nusieve GTG agarose gels. Lane 1 (C76), lane 2 (5mC76) and lane 3 (5hmC76) show clean single bands for each of the PCR products. After BstUI digestion, C76 (lane 4) was fully digested, whereas samples in lane 5 (5mC76) and lane 6 (5hmC76) resisted digestion. ( C ) Analysis of PCR products C76, 5mC76 and 5hmC76 by cleavage with different methylation-sensitive restriction enzymes. C76 (lanes 1, 4, 7 and 10), 5mC76 (lanes 2, 5, 8 and 11) and 5hmC76 (lanes 3, 6, 9 and 12) were left untreated (lanes 1–3) or were incubated with MluI (lanes 4–6), NruI (lanes 7–9) or HhaI (lanes 9–12).

Techniques Used: Modification, Sequencing, Synthesized, Polymerase Chain Reaction, Methylation, Electrophoresis, Incubation

DNA immunoprecipitation with anti-5mC antibody. Immunoprecipitation with an antibody against 5-methylcytidine was carried out to test the antibody’s affinity towards modified cytosines. The levels of immunocaptured 76-mers were measured using liquid scintillation counting. As a control, normal mouse IgG was used for immunoprecipitation. The oligonucleotides C76, 5mC76 and 5hmC76 were synthesized by PCR and contain C, 5mC or 5hmC at three BstUI sites as schematically indicated at the top of the Figure (see Figure 1 for sequence). Oligonucleotides 5mC5hmC76 and 5mC76a were prepared by chemical synthesis and contain 5hmC or C at the central BstUI site. Experiments were done in triplicates and the standard deviation is shown.
Figure Legend Snippet: DNA immunoprecipitation with anti-5mC antibody. Immunoprecipitation with an antibody against 5-methylcytidine was carried out to test the antibody’s affinity towards modified cytosines. The levels of immunocaptured 76-mers were measured using liquid scintillation counting. As a control, normal mouse IgG was used for immunoprecipitation. The oligonucleotides C76, 5mC76 and 5hmC76 were synthesized by PCR and contain C, 5mC or 5hmC at three BstUI sites as schematically indicated at the top of the Figure (see Figure 1 for sequence). Oligonucleotides 5mC5hmC76 and 5mC76a were prepared by chemical synthesis and contain 5hmC or C at the central BstUI site. Experiments were done in triplicates and the standard deviation is shown.

Techniques Used: Immunoprecipitation, Modification, Synthesized, Polymerase Chain Reaction, Sequencing, Standard Deviation

13) Product Images from "Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27kip1"

Article Title: Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27kip1

Journal: Respiratory Research

doi: 10.1186/1465-9921-8-77

Effect of BPS and 8-Br-cAMP on p27 kip1 mRNA stability during hypoxia . Cultured HPASMC were exposed to 21% or 2% oxygen concentrations with or without 10 μM of BPS or 1 mM 8-Br-cAMP for indicated periods. The p27 kip1 mRNA stability was measured after adding 400 nM of Act D using Real-time RT-PCR using LightCycler™. Degradation of p27 kip1 mRNA was significantly suppressed by BPS and 8-Br-cAMP under both normoxic and moderately hypoxic conditions, and mRNA stability was slightly decreased by moderate hypoxia. Graphs show % maximal p27 kip1 mRNA expression. Line with solid circles, 21% oxygen; dotted line with open circles, 2% oxygen; line with solid squares, 21% oxygen and BPS; line with solid triangles, 21% oxygen and 8-Br-cAMP; dotted line with open squares, 2% oxygen and BPS; dotted line with open triangle, 2% oxygen and 8-Br-cAMP. Data are expressed as means ± SE (n = 6). *P
Figure Legend Snippet: Effect of BPS and 8-Br-cAMP on p27 kip1 mRNA stability during hypoxia . Cultured HPASMC were exposed to 21% or 2% oxygen concentrations with or without 10 μM of BPS or 1 mM 8-Br-cAMP for indicated periods. The p27 kip1 mRNA stability was measured after adding 400 nM of Act D using Real-time RT-PCR using LightCycler™. Degradation of p27 kip1 mRNA was significantly suppressed by BPS and 8-Br-cAMP under both normoxic and moderately hypoxic conditions, and mRNA stability was slightly decreased by moderate hypoxia. Graphs show % maximal p27 kip1 mRNA expression. Line with solid circles, 21% oxygen; dotted line with open circles, 2% oxygen; line with solid squares, 21% oxygen and BPS; line with solid triangles, 21% oxygen and 8-Br-cAMP; dotted line with open squares, 2% oxygen and BPS; dotted line with open triangle, 2% oxygen and 8-Br-cAMP. Data are expressed as means ± SE (n = 6). *P

Techniques Used: Cell Culture, Activated Clotting Time Assay, Quantitative RT-PCR, Expressing

Effects of BPS and 8-Br-cAMPon p27 kip1 mRNA expression during hypoxia . Cultured HPASMC were exposed to 21% or 2% oxygen concentrations with or without 10 μM of BPS or 1 mM 8-Br-cAMP for indicated periods. Expression of p27 kip1 mRNA was measured using Real-time RT-PCR using LightCycler™. (a) BPS suppressed p27 kip1 mRNA reduction under both normoxic and hypoxic conditions. Expression of p27 kip1 mRNA between normoxic and hypoxic conditions did not significantly change. Graph shows ratio of p27 kip1 to β-actin mRNA expression. Open and solid bars, 21% and 2% oxygen, respectively. Data are expressed as means ± SE (n = 6). *P
Figure Legend Snippet: Effects of BPS and 8-Br-cAMPon p27 kip1 mRNA expression during hypoxia . Cultured HPASMC were exposed to 21% or 2% oxygen concentrations with or without 10 μM of BPS or 1 mM 8-Br-cAMP for indicated periods. Expression of p27 kip1 mRNA was measured using Real-time RT-PCR using LightCycler™. (a) BPS suppressed p27 kip1 mRNA reduction under both normoxic and hypoxic conditions. Expression of p27 kip1 mRNA between normoxic and hypoxic conditions did not significantly change. Graph shows ratio of p27 kip1 to β-actin mRNA expression. Open and solid bars, 21% and 2% oxygen, respectively. Data are expressed as means ± SE (n = 6). *P

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

14) Product Images from "Loss-of-Function Mutations in PTPN11 Cause Metachondromatosis, but Not Ollier Disease or Maffucci Syndrome"

Article Title: Loss-of-Function Mutations in PTPN11 Cause Metachondromatosis, but Not Ollier Disease or Maffucci Syndrome

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002050

PTPN11 mutations identified in MC participants. (A) Schematic of the exonic structure of PTPN11 (above) and the corresponding protein structure of SHP2 (below). The locations of mutations that were identified in MC are indicated with black lines. Predicted protein changes are indicated for the nonsense (blue) and frameshift (red) mutations, while the cDNA designation is indicated for the splice-site mutations (green). (B) Log2 values of the number of Illumina reads obtained per 50 bp window in participant S, divided by the average number of reads obtained in other participants whose DNA was captured simultaneously using the second capture array. Shown are all 50 bp windows spanning regions of PTPN11 targeted by the array, with the corresponding exonic structure of PTPN11 shown below. The red bar indicates a region spanning exon 7, in which the average log2 value is approximately −1, suggesting a heterozygous deletion. PCR amplification and sequencing of the breakpoint, using primers on either end of the deletion, indicate that 14,629 bp of sequence have been deleted and replaced with a single CA dinucleotide.
Figure Legend Snippet: PTPN11 mutations identified in MC participants. (A) Schematic of the exonic structure of PTPN11 (above) and the corresponding protein structure of SHP2 (below). The locations of mutations that were identified in MC are indicated with black lines. Predicted protein changes are indicated for the nonsense (blue) and frameshift (red) mutations, while the cDNA designation is indicated for the splice-site mutations (green). (B) Log2 values of the number of Illumina reads obtained per 50 bp window in participant S, divided by the average number of reads obtained in other participants whose DNA was captured simultaneously using the second capture array. Shown are all 50 bp windows spanning regions of PTPN11 targeted by the array, with the corresponding exonic structure of PTPN11 shown below. The red bar indicates a region spanning exon 7, in which the average log2 value is approximately −1, suggesting a heterozygous deletion. PCR amplification and sequencing of the breakpoint, using primers on either end of the deletion, indicate that 14,629 bp of sequence have been deleted and replaced with a single CA dinucleotide.

Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

Loss of the wild-type PTPN11 allele in the cartilage of two exostoses. (A) Electropherograms of PCR amplified template DNA that had been extracted from whole blood, a section of an exostosis, or the cartilage core of the same exostosis. Exostoses were available from patients A-IV-5 and A-IV-8. The site of the 5 bp deletion in exon 4 of PTPN11 in both patients is indicated with a box. Note that that the heights of the peaks corresponding to the mutant sequence are markedly reduced in amplimers from the cartilage-core compared to amplimers from blood or from a section that contains cartilage, bone and fibrous tissue. This is consistent with loss-of-heterozygosity in the cartilage component of the exostoses. (B) Electropherograms of PCR amplified template DNA extracted from blood from participants A-III-9, A-III-10 and A-IV-5, as well as DNA extracted from the cartilage core of the exostosis from participant A-IV-5 shown in (A). Blood DNA electropherograms indicate that participants A-III-9 and A-IV-10 are heterozygous at a position (asterisk) in intron 11 of PTPN11 . This is the site of a known common polymorphism (rs41279092). Exostosis cartilage DNA electropherograms have a reduced adenine peak height at this position. This suggests that the wild-type PTPN11 allele inherited from the unaffected parent (A-III-9), which carries an adenine at this position, has been lost in cells that contribute to formation of the exostosis' cartilage core.
Figure Legend Snippet: Loss of the wild-type PTPN11 allele in the cartilage of two exostoses. (A) Electropherograms of PCR amplified template DNA that had been extracted from whole blood, a section of an exostosis, or the cartilage core of the same exostosis. Exostoses were available from patients A-IV-5 and A-IV-8. The site of the 5 bp deletion in exon 4 of PTPN11 in both patients is indicated with a box. Note that that the heights of the peaks corresponding to the mutant sequence are markedly reduced in amplimers from the cartilage-core compared to amplimers from blood or from a section that contains cartilage, bone and fibrous tissue. This is consistent with loss-of-heterozygosity in the cartilage component of the exostoses. (B) Electropherograms of PCR amplified template DNA extracted from blood from participants A-III-9, A-III-10 and A-IV-5, as well as DNA extracted from the cartilage core of the exostosis from participant A-IV-5 shown in (A). Blood DNA electropherograms indicate that participants A-III-9 and A-IV-10 are heterozygous at a position (asterisk) in intron 11 of PTPN11 . This is the site of a known common polymorphism (rs41279092). Exostosis cartilage DNA electropherograms have a reduced adenine peak height at this position. This suggests that the wild-type PTPN11 allele inherited from the unaffected parent (A-III-9), which carries an adenine at this position, has been lost in cells that contribute to formation of the exostosis' cartilage core.

Techniques Used: Polymerase Chain Reaction, Amplification, Mutagenesis, Sequencing

15) Product Images from "Induction of exportin-5 expression during melanoma development supports the cellular behavior of human malignant melanoma cells"

Article Title: Induction of exportin-5 expression during melanoma development supports the cellular behavior of human malignant melanoma cells

Journal: Oncotarget

doi: 10.18632/oncotarget.11410

Elevated XPO5 mRNA expression and stability in malignant melanoma A. qRT-PCRs showed an increase in expression of XPO5 mRNA during melanoma progression in primary and metastatic melanoma tissues (n=4) and cell lines (n=13) compared with NHEMs (n=15). B. XPO5 mRNA expression decreased after treatment with the MEK inhibitors PD98059 and U0126 in comparison to DMSO-treated cells (n=10). C. The stability of XPO5 mRNA was determined by qRT-PCR after treatment of NHEMs and melanoma cell lines (MM) with alpha-amanitin for 0, 16 and 24 hours. The remaining XPO5 mRNA level in the melanoma cell lines (solid line) was significantly different to that of the NHEMs (dashed line) after the 24 h treatment (n=3). D. The relative distribution of the rs11077 genotypes in melanoma patients (n=20) versus the healthy control group (n-21). In the control group, the values are equivalent to the distribution in the Caucasian population. In melanoma patients, half of the patients had the C/C variant SNP (50%). E. Relative XPO5 mRNA expression compared to NHEM in the homozygous rs11077 genotypes A/A and C/C.
Figure Legend Snippet: Elevated XPO5 mRNA expression and stability in malignant melanoma A. qRT-PCRs showed an increase in expression of XPO5 mRNA during melanoma progression in primary and metastatic melanoma tissues (n=4) and cell lines (n=13) compared with NHEMs (n=15). B. XPO5 mRNA expression decreased after treatment with the MEK inhibitors PD98059 and U0126 in comparison to DMSO-treated cells (n=10). C. The stability of XPO5 mRNA was determined by qRT-PCR after treatment of NHEMs and melanoma cell lines (MM) with alpha-amanitin for 0, 16 and 24 hours. The remaining XPO5 mRNA level in the melanoma cell lines (solid line) was significantly different to that of the NHEMs (dashed line) after the 24 h treatment (n=3). D. The relative distribution of the rs11077 genotypes in melanoma patients (n=20) versus the healthy control group (n-21). In the control group, the values are equivalent to the distribution in the Caucasian population. In melanoma patients, half of the patients had the C/C variant SNP (50%). E. Relative XPO5 mRNA expression compared to NHEM in the homozygous rs11077 genotypes A/A and C/C.

Techniques Used: Expressing, Quantitative RT-PCR, Variant Assay

16) Product Images from "Sp1 and Sp3 regulate basal transcription of the human APOBEC3G gene"

Article Title: Sp1 and Sp3 regulate basal transcription of the human APOBEC3G gene

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm340

5′-RACE analysis of the A3G cDNA. Agarose gel electrophoresis of size marker and A3G 5′-RACE products after nested PCR with primer RACE-APO3Gnest (see Figure 1 for primer details). Arrowheads indicate the three resulting DNA bands which were cloned and sequenced.
Figure Legend Snippet: 5′-RACE analysis of the A3G cDNA. Agarose gel electrophoresis of size marker and A3G 5′-RACE products after nested PCR with primer RACE-APO3Gnest (see Figure 1 for primer details). Arrowheads indicate the three resulting DNA bands which were cloned and sequenced.

Techniques Used: Agarose Gel Electrophoresis, Marker, Nested PCR, Clone Assay

Sp1 and Sp3 bind to the GC-box present in the A3G promoter. ( A ) Nuclear extracts of A3.01 T cells were incubated with a 32 P-labeled commercial Sp1 oligonucleotide probe (Sp1cons) or labeled probes homologous to the unmodified or mutated E2 region (see Figure 1 ) of the A3G promoter (APO-Sp1/3 and APO-Sp1/3mut). Protein–DNA complexes were separated by polyacrylamide electrophoresis and detected by autoradiography. EMSA was performed with a 1- or 30-fold molar excess of unlabeled APO-Sp1/3 probe (competitor, lanes 6 and 7) or APO-Sp1/3mut probe (competitor mut., lanes 12 and 13). ( B ) Sp1- and Sp3-specific antibodies were added to the EMSA reactions resulting in a supershift (ss) of the respective antibody–protein–oligo complexes (lanes 3, 4, 7, 8, 11, 12). ( C ) ChIP assay was performed with DNA from A3.01 T cells. Immunoprecipitation was performed with antibodies against Sp1, Sp3 or actin. PCR primer pairs specific for the A3G promoter (upper panel) or the A3G gene (lower panel) were used. As positive controls, the sheared and cross-linked DNA before the immunoprecipitation step (input) or a plasmid carrying the target sequence (plasmid) was used as template.
Figure Legend Snippet: Sp1 and Sp3 bind to the GC-box present in the A3G promoter. ( A ) Nuclear extracts of A3.01 T cells were incubated with a 32 P-labeled commercial Sp1 oligonucleotide probe (Sp1cons) or labeled probes homologous to the unmodified or mutated E2 region (see Figure 1 ) of the A3G promoter (APO-Sp1/3 and APO-Sp1/3mut). Protein–DNA complexes were separated by polyacrylamide electrophoresis and detected by autoradiography. EMSA was performed with a 1- or 30-fold molar excess of unlabeled APO-Sp1/3 probe (competitor, lanes 6 and 7) or APO-Sp1/3mut probe (competitor mut., lanes 12 and 13). ( B ) Sp1- and Sp3-specific antibodies were added to the EMSA reactions resulting in a supershift (ss) of the respective antibody–protein–oligo complexes (lanes 3, 4, 7, 8, 11, 12). ( C ) ChIP assay was performed with DNA from A3.01 T cells. Immunoprecipitation was performed with antibodies against Sp1, Sp3 or actin. PCR primer pairs specific for the A3G promoter (upper panel) or the A3G gene (lower panel) were used. As positive controls, the sheared and cross-linked DNA before the immunoprecipitation step (input) or a plasmid carrying the target sequence (plasmid) was used as template.

Techniques Used: Incubation, Labeling, Electrophoresis, Autoradiography, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Sequencing

17) Product Images from "Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation"

Article Title: Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.141222198

RT-PCR products of error-prone DNA polymerases at 48 h. cDNA from cells that were untreated or treated under various conditions (+ anti-IgM, + Hut 78, or + IgM + HuT 78) for 0, 12, 24, and 48 h were used in RT-PCR. Stock dilutions (1:10, 1:20, and 1:40) of the cDNA were made and used in the amplifications. For each time point and condition, undiluted cDNA and the aforementioned dilutions were used to detect pols ζ, η, and ι, and GAPDH. No PCR products were detected in the no-RT control cDNAs for each condition (data not shown).
Figure Legend Snippet: RT-PCR products of error-prone DNA polymerases at 48 h. cDNA from cells that were untreated or treated under various conditions (+ anti-IgM, + Hut 78, or + IgM + HuT 78) for 0, 12, 24, and 48 h were used in RT-PCR. Stock dilutions (1:10, 1:20, and 1:40) of the cDNA were made and used in the amplifications. For each time point and condition, undiluted cDNA and the aforementioned dilutions were used to detect pols ζ, η, and ι, and GAPDH. No PCR products were detected in the no-RT control cDNAs for each condition (data not shown).

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

Semiquantitation of RT-PCR products of error-prone DNA polymerases in BL2. RT-PCR was performed for pols ζ, η, and ι at 0, 12, 24, and 48 h after various conditions. For each time point and condition, RT-PCR was performed for the housekeeping gene, GAPDH . The OD of each band for the polymerases was measured and normalized against the OD of GAPDH at the respective time point and condition. The PCR products from the 1:10 and 1:20 dilutions of the template cDNA were normalized individually to GAPDH. The normalized values of the PCR products of the 1:20 dilution were doubled and then averaged with the 1:10 PCR products. The average is shown in the bar graph, and the error between the two determinations is depicted by the error bars.
Figure Legend Snippet: Semiquantitation of RT-PCR products of error-prone DNA polymerases in BL2. RT-PCR was performed for pols ζ, η, and ι at 0, 12, 24, and 48 h after various conditions. For each time point and condition, RT-PCR was performed for the housekeeping gene, GAPDH . The OD of each band for the polymerases was measured and normalized against the OD of GAPDH at the respective time point and condition. The PCR products from the 1:10 and 1:20 dilutions of the template cDNA were normalized individually to GAPDH. The normalized values of the PCR products of the 1:20 dilution were doubled and then averaged with the 1:10 PCR products. The average is shown in the bar graph, and the error between the two determinations is depicted by the error bars.

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

18) Product Images from "Constitutive translation of human α-synuclein is mediated by the 5′-untranslated region"

Article Title: Constitutive translation of human α-synuclein is mediated by the 5′-untranslated region

Journal: Open Biology

doi: 10.1098/rsob.160022

The SNCA 5′-UTR enhances luciferase reporter activity. ( a ) Schematic of the psiCHECK2 bicistronic expression constructs. The SNCA and GAPDH 5′-UTR cDNAs were inserted upstream of Renilla luciferase gene in the psiCHECK2 dual reporter vector. ( b,c ) Empty (no insert) control plasmid and plasmids containing the SNCA or GAPDH 5′-UTR were transiently transfected into HEK-293 cells. Forty-eight hours later, cells were harvested and dual luciferase assays was performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of Renilla to firefly (Rluc/Fluc) activity. Relative mRNA levels were determined by real-time RT-PCR. The results are expressed as the ratio of Renilla to firefly expression. Note that only the psiCHECK2 construct that incorporates the SNCA 5′-UTR induced luciferase protein levels. The figures represent averages and standard errors of four independent experiments. * p
Figure Legend Snippet: The SNCA 5′-UTR enhances luciferase reporter activity. ( a ) Schematic of the psiCHECK2 bicistronic expression constructs. The SNCA and GAPDH 5′-UTR cDNAs were inserted upstream of Renilla luciferase gene in the psiCHECK2 dual reporter vector. ( b,c ) Empty (no insert) control plasmid and plasmids containing the SNCA or GAPDH 5′-UTR were transiently transfected into HEK-293 cells. Forty-eight hours later, cells were harvested and dual luciferase assays was performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of Renilla to firefly (Rluc/Fluc) activity. Relative mRNA levels were determined by real-time RT-PCR. The results are expressed as the ratio of Renilla to firefly expression. Note that only the psiCHECK2 construct that incorporates the SNCA 5′-UTR induced luciferase protein levels. The figures represent averages and standard errors of four independent experiments. * p

Techniques Used: Luciferase, Activity Assay, Expressing, Construct, Plasmid Preparation, Transfection, Quantitative RT-PCR

19) Product Images from "A collective form of cell death requires homeodomain interacting protein kinase"

Article Title: A collective form of cell death requires homeodomain interacting protein kinase

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200702125

HIPK is essential for normal PCD in the wing epithelium. The HIPK locus is depicted (A) with the original insertion, l(3)S134313. Other transposons, f03158 and d10792 , were used to generate a deletion HIPK D1 (C), which removes 92% of the HIPK coding region by replacing residues 1–1243 of the HIPK coding sequences with white+ marker gene. Primer sets A-B and C-D (C) were used to identify recombination events by genomic PCR. Deletions were verified in panel B using two additional primer sets, which resulted in a novel PCR product (primers A–D) and a negative PCR result (primers E-F) in homozygous mutant DNA. rp49 represents a positive control. Wings mosaic for both HIPK l(3)S134313 (D and F) and HIPK D1 (E and G) were normal at eclosion, but upon aging showed severe blemishing and persisting cell phenotypes (D–G), with HIPK D1 wings exhibiting additional persisting DsRed cells (G). Note that the progressive blemishing phenotypes were not seen in parental strains, including those used to produce FRT recombinants.
Figure Legend Snippet: HIPK is essential for normal PCD in the wing epithelium. The HIPK locus is depicted (A) with the original insertion, l(3)S134313. Other transposons, f03158 and d10792 , were used to generate a deletion HIPK D1 (C), which removes 92% of the HIPK coding region by replacing residues 1–1243 of the HIPK coding sequences with white+ marker gene. Primer sets A-B and C-D (C) were used to identify recombination events by genomic PCR. Deletions were verified in panel B using two additional primer sets, which resulted in a novel PCR product (primers A–D) and a negative PCR result (primers E-F) in homozygous mutant DNA. rp49 represents a positive control. Wings mosaic for both HIPK l(3)S134313 (D and F) and HIPK D1 (E and G) were normal at eclosion, but upon aging showed severe blemishing and persisting cell phenotypes (D–G), with HIPK D1 wings exhibiting additional persisting DsRed cells (G). Note that the progressive blemishing phenotypes were not seen in parental strains, including those used to produce FRT recombinants.

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

20) Product Images from "Bioactive Dietary VDR Ligands Regulate Genes Encoding Biomarkers of Skin Repair That Are Associated with Risk for Psoriasis"

Article Title: Bioactive Dietary VDR Ligands Regulate Genes Encoding Biomarkers of Skin Repair That Are Associated with Risk for Psoriasis

Journal: Nutrients

doi: 10.3390/nu10020174

Regulation of NURR1 ( NR4A2 ) mRNA by 1,25D. ( A ) Response of NURR1 mRNA to 1,25D and DHA in HEKn cells. Cells were plated as described in Methods and dosed with the indicated concentrations of 1,25D or DHA for 22–24 h. Total RNA and first strand cDNA were then prepared, and real-time qPCR was performed using primers to human NURR1 as described in Methods. Error bars represent STDEV of triplicate real-time PCR wells from each of two independent experiments. ( B ) A similar experiment to ( A ), but performed using increasing doses of 1,25D in rat UMR-106 cultures. Results are means of three independent experiments ± STDEV, * p
Figure Legend Snippet: Regulation of NURR1 ( NR4A2 ) mRNA by 1,25D. ( A ) Response of NURR1 mRNA to 1,25D and DHA in HEKn cells. Cells were plated as described in Methods and dosed with the indicated concentrations of 1,25D or DHA for 22–24 h. Total RNA and first strand cDNA were then prepared, and real-time qPCR was performed using primers to human NURR1 as described in Methods. Error bars represent STDEV of triplicate real-time PCR wells from each of two independent experiments. ( B ) A similar experiment to ( A ), but performed using increasing doses of 1,25D in rat UMR-106 cultures. Results are means of three independent experiments ± STDEV, * p

Techniques Used: Real-time Polymerase Chain Reaction

( A ) Regulation of LCE3A, LCE3D , and LCE3E mRNA by curcumin (Crc). HEKn cells were treated with ETOH vehicle (negative control) or curcumin (at 6.7 or 10 μM concentration). Bar graphs show real-time PCR results, which are the average of four independent experiments ± STDEV. An asterisk denotes ligand-treated averages that are significantly different from ethanol controls as determined by two-tailed Student’s t -test, * p
Figure Legend Snippet: ( A ) Regulation of LCE3A, LCE3D , and LCE3E mRNA by curcumin (Crc). HEKn cells were treated with ETOH vehicle (negative control) or curcumin (at 6.7 or 10 μM concentration). Bar graphs show real-time PCR results, which are the average of four independent experiments ± STDEV. An asterisk denotes ligand-treated averages that are significantly different from ethanol controls as determined by two-tailed Student’s t -test, * p

Techniques Used: Negative Control, Concentration Assay, Real-time Polymerase Chain Reaction, Two Tailed Test

( A ) Regulation of Jun B mRNA by DHA. HEKn cells were treated with ethanol vehicle (control), or with either 10 μM DHA or 20 μM DHA. RNA isolation, synthesis of first strand DNA, and real-time PCR are described in Methods. Results are means from three independent experiments ± STDEV. The double asterisk (**) denotes an average that is statistically significant by Student’s t -test from the ethanol control, p
Figure Legend Snippet: ( A ) Regulation of Jun B mRNA by DHA. HEKn cells were treated with ethanol vehicle (control), or with either 10 μM DHA or 20 μM DHA. RNA isolation, synthesis of first strand DNA, and real-time PCR are described in Methods. Results are means from three independent experiments ± STDEV. The double asterisk (**) denotes an average that is statistically significant by Student’s t -test from the ethanol control, p

Techniques Used: Isolation, Real-time Polymerase Chain Reaction

21) Product Images from "Type I IFN induced IL1-Ra expression in hepatocytes is mediated by activating STAT6 through the formation of STAT2: STAT6 heterodimer"

Article Title: Type I IFN induced IL1-Ra expression in hepatocytes is mediated by activating STAT6 through the formation of STAT2: STAT6 heterodimer

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2008.00143.x

Treatment of HuH7 cells with IFNα, IFNβ, and IL-4 significantly enhanced STAT6 binding to CD23 and IL-1Ra promoter. Extracts were prepared from Huh7 cells with or without a 30-min stimulation at 37°C with IL-4 (10 ng/ml) (lane 1 and 5), phosphate buffered saline (lane 2 and 6;red lines for ChIP), IFNα (400 IU/ml;blue lines for ChIP) (lane 3 and 7; blue line for ChIP), and IFNβ (400 IU/ml; green lines for ChIP) (lane 4 and 8). The nuclear extracts were examined by electrophoretic mobility shift assay (EMSA) using either biotin-labelled CD23 ( A ) and IL-1Ra ( B ) GAS probes alone or together with 100-fold molar excess of non-biotin labelled probes. The antibodies against STAT6 inhibited STAT6 binding to IL-1Ra GAS probes ( C ). Activated STAT6 is bound to the IL-1Ra promoter in native chromatin determined by ChIP assays ( D ). DNA fragments recovered from ChIP assays with anti-STAT6 (solid lines) or anti-GAPDH (dashed lines) were analysed by quantitative PCR using Roche universal probe 87. The Cp values were shown in parentheses.
Figure Legend Snippet: Treatment of HuH7 cells with IFNα, IFNβ, and IL-4 significantly enhanced STAT6 binding to CD23 and IL-1Ra promoter. Extracts were prepared from Huh7 cells with or without a 30-min stimulation at 37°C with IL-4 (10 ng/ml) (lane 1 and 5), phosphate buffered saline (lane 2 and 6;red lines for ChIP), IFNα (400 IU/ml;blue lines for ChIP) (lane 3 and 7; blue line for ChIP), and IFNβ (400 IU/ml; green lines for ChIP) (lane 4 and 8). The nuclear extracts were examined by electrophoretic mobility shift assay (EMSA) using either biotin-labelled CD23 ( A ) and IL-1Ra ( B ) GAS probes alone or together with 100-fold molar excess of non-biotin labelled probes. The antibodies against STAT6 inhibited STAT6 binding to IL-1Ra GAS probes ( C ). Activated STAT6 is bound to the IL-1Ra promoter in native chromatin determined by ChIP assays ( D ). DNA fragments recovered from ChIP assays with anti-STAT6 (solid lines) or anti-GAPDH (dashed lines) were analysed by quantitative PCR using Roche universal probe 87. The Cp values were shown in parentheses.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Electrophoretic Mobility Shift Assay, Real-time Polymerase Chain Reaction

22) Product Images from "Duplex Scorpion primers in SNP analysis and FRET applications"

Article Title: Duplex Scorpion primers in SNP analysis and FRET applications

Journal: Nucleic Acids Research

doi:

( A ) PCR amplification with FRET duplex Scorpions W-006/W-007, W-009/W-010 and W-011/W-012 (proportional mode). ROX fluorescence was monitored. ( B ) Allelic discrimination with FRET duplex Scorpion W-006/W-007. ROX fluorescence was monitored in channel 2 of the LightCycler (arithmetic mode).
Figure Legend Snippet: ( A ) PCR amplification with FRET duplex Scorpions W-006/W-007, W-009/W-010 and W-011/W-012 (proportional mode). ROX fluorescence was monitored. ( B ) Allelic discrimination with FRET duplex Scorpion W-006/W-007. ROX fluorescence was monitored in channel 2 of the LightCycler (arithmetic mode).

Techniques Used: Polymerase Chain Reaction, Amplification, Fluorescence

23) Product Images from "Target Motifs Affecting Natural Immunity by a Constitutive CRISPR-Cas System in Escherichia coli"

Article Title: Target Motifs Affecting Natural Immunity by a Constitutive CRISPR-Cas System in Escherichia coli

Journal: PLoS ONE

doi: 10.1371/journal.pone.0050797

Expression of cas1 and csy1 genes revealed by RT-PCR. Agarose gel electrophoresis of PCR products obtained using as template total DNA (lanes 1 to 3), cDNA (lanes 5 to 7) or RNA (lanes 8 to 10) of LF82 strain grown in LB medium at logarithmic phase (results of samples from stationary phase cultures were similar and are not shown). In addition to cas 1 (lanes 1, 5 and 8) and csy1 (lanes 2, 6 and 9) the highly expressed tufB transcript (lanes 3, 7 and 10) was probed as a control of DNA contamination in RNA samples. A molecular weight marker is included (lane 4) for fragment size estimation.
Figure Legend Snippet: Expression of cas1 and csy1 genes revealed by RT-PCR. Agarose gel electrophoresis of PCR products obtained using as template total DNA (lanes 1 to 3), cDNA (lanes 5 to 7) or RNA (lanes 8 to 10) of LF82 strain grown in LB medium at logarithmic phase (results of samples from stationary phase cultures were similar and are not shown). In addition to cas 1 (lanes 1, 5 and 8) and csy1 (lanes 2, 6 and 9) the highly expressed tufB transcript (lanes 3, 7 and 10) was probed as a control of DNA contamination in RNA samples. A molecular weight marker is included (lane 4) for fragment size estimation.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Molecular Weight, Marker

24) Product Images from "Forensic pregnancy diagnostics with placental mRNA markers"

Article Title: Forensic pregnancy diagnostics with placental mRNA markers

Journal: International Journal of Legal Medicine

doi: 10.1007/s00414-008-0315-6

Agarose gel electrophoresis with ethidium bromide staining of RT-PCR reactions with RNA from whole blood of eight different pregnant women in their 13th until 37th week of pregnancy ( lanes 1 – 8 ) and four different non-pregnant women ( lanes 9 – 12 ). a hPL mRNA amplifications (expected product size: 97 bp). b GAPDH mRNA amplifications (expected product size: 260 bp). Lane M corresponds to a 50-base pair molecular size ladder and lane 13 corresponds to no template control reactions
Figure Legend Snippet: Agarose gel electrophoresis with ethidium bromide staining of RT-PCR reactions with RNA from whole blood of eight different pregnant women in their 13th until 37th week of pregnancy ( lanes 1 – 8 ) and four different non-pregnant women ( lanes 9 – 12 ). a hPL mRNA amplifications (expected product size: 97 bp). b GAPDH mRNA amplifications (expected product size: 260 bp). Lane M corresponds to a 50-base pair molecular size ladder and lane 13 corresponds to no template control reactions

Techniques Used: Agarose Gel Electrophoresis, Staining, Reverse Transcription Polymerase Chain Reaction

25) Product Images from "Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)-related protein Sli15 during chromosome segregation"

Article Title: Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)-related protein Sli15 during chromosome segregation

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200105029

Association of Ipl1 and Sli15 with centromeric DNA. Extracts prepared from formaldehyde-fixed yeast cells (CCY776-5D) that did not (untagged) or did express HA–Ipl1 (pCC1128) or Sli15–Myc (pCC1193) were prepared. These extracts were left untreated (WCE), mock-precipitated without antibodies (No Ab), or immunoprecipitated with anti-HA (α HA) or anti-Myc (α Myc) antibodies. PCR was performed to amplify DNA fragments from WCE or precipitated samples, using primers that flank CEN3 or CEN16, or primers from a region located ∼1 kb from CEN16.
Figure Legend Snippet: Association of Ipl1 and Sli15 with centromeric DNA. Extracts prepared from formaldehyde-fixed yeast cells (CCY776-5D) that did not (untagged) or did express HA–Ipl1 (pCC1128) or Sli15–Myc (pCC1193) were prepared. These extracts were left untreated (WCE), mock-precipitated without antibodies (No Ab), or immunoprecipitated with anti-HA (α HA) or anti-Myc (α Myc) antibodies. PCR was performed to amplify DNA fragments from WCE or precipitated samples, using primers that flank CEN3 or CEN16, or primers from a region located ∼1 kb from CEN16.

Techniques Used: Immunoprecipitation, Polymerase Chain Reaction

26) Product Images from "Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms"

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms

Journal: Retrovirology

doi: 10.1186/1742-4690-2-64

DNA methylation of provirus is not associated with methylated CpG sites in the genome. Integration sites of HTLV-I provirus in leukemic cells have been determined by inverse PCR, and then DNA methylation in genome has been analyzed by sodium bisulfite sequencing. DNA methylation of 5'-LTR was also analyzed by sodium bisulfite sequencing method. Vertical bars represent CpG sites. Open circle indicates unmethylated CpG site, and closed one means methylated CpG site. N: normal PBMCs from non-carrier donor.
Figure Legend Snippet: DNA methylation of provirus is not associated with methylated CpG sites in the genome. Integration sites of HTLV-I provirus in leukemic cells have been determined by inverse PCR, and then DNA methylation in genome has been analyzed by sodium bisulfite sequencing. DNA methylation of 5'-LTR was also analyzed by sodium bisulfite sequencing method. Vertical bars represent CpG sites. Open circle indicates unmethylated CpG site, and closed one means methylated CpG site. N: normal PBMCs from non-carrier donor.

Techniques Used: DNA Methylation Assay, Methylation, Inverse PCR, Methylation Sequencing

DNA methylation and histone modifications in fresh ATL cases. A. The relationships among DNA methylation, tax gene expression and histone modification in 5'-LTR were analyzed in three ATL cases. Cases 1 and 3 have one copy of the complete HTLV-I provirus, while Case 2 has a defective provirus that lacks part of the pol gene. DNA methylation was analyzed by COBRA. The tax gene transcripts could be detected in Case 1, but not in Cases 2 or 3, by RT-PCR. ChIP assays were also performed using primers for 5'-LTR to analyze acetylation of histone H3 (Ac-H3) and H4 (Ac-H4). W.C.E.: whole cell extract. B. Recovery of tax gene expression ex vivo . The PBMCs isolated from Case 3 were immediately cultured ex vivo for several hours and tested the transcription of tax mRNA by RT-PCR.
Figure Legend Snippet: DNA methylation and histone modifications in fresh ATL cases. A. The relationships among DNA methylation, tax gene expression and histone modification in 5'-LTR were analyzed in three ATL cases. Cases 1 and 3 have one copy of the complete HTLV-I provirus, while Case 2 has a defective provirus that lacks part of the pol gene. DNA methylation was analyzed by COBRA. The tax gene transcripts could be detected in Case 1, but not in Cases 2 or 3, by RT-PCR. ChIP assays were also performed using primers for 5'-LTR to analyze acetylation of histone H3 (Ac-H3) and H4 (Ac-H4). W.C.E.: whole cell extract. B. Recovery of tax gene expression ex vivo . The PBMCs isolated from Case 3 were immediately cultured ex vivo for several hours and tested the transcription of tax mRNA by RT-PCR.

Techniques Used: DNA Methylation Assay, Expressing, Modification, Combined Bisulfite Restriction Analysis Assay, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Ex Vivo, Isolation, Cell Culture

DNA methylation in ATL cell lines, HTLV-I carriers and ATL cases. The tax gene transcription in ATL cell lines was studied by RT-PCR (A), and the expression of GAPDH gene has been used as a control. DNA methylation throughout the HTLV-I provirus was studied by COBRA in tax gene-expressing (B) and non-expressing cell lines (C). Furthermore, DNA methylation was also analyzed in 20 carriers and 20 ATL cases by COBRA, and representative patterns of DNA methylation are shown in D. The number of HTLV-I provirus has been analyzed by Southern blot method, and shown in the parenthesis (B, C and D). Each bar indicates the extent of DNA methylation that was calculated by COBRA.
Figure Legend Snippet: DNA methylation in ATL cell lines, HTLV-I carriers and ATL cases. The tax gene transcription in ATL cell lines was studied by RT-PCR (A), and the expression of GAPDH gene has been used as a control. DNA methylation throughout the HTLV-I provirus was studied by COBRA in tax gene-expressing (B) and non-expressing cell lines (C). Furthermore, DNA methylation was also analyzed in 20 carriers and 20 ATL cases by COBRA, and representative patterns of DNA methylation are shown in D. The number of HTLV-I provirus has been analyzed by Southern blot method, and shown in the parenthesis (B, C and D). Each bar indicates the extent of DNA methylation that was calculated by COBRA.

Techniques Used: DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Combined Bisulfite Restriction Analysis Assay, Southern Blot

Comparison of the DNA methylation in carriers and ATL cases. A. DNA methylation at eight different regions in the HTLV-I provirus was compared between carriers (C) and ATL cases (A). DNA methylation was quantified by COBRA in 20 carriers and 20 ATL cases. Each sample was analyzed three times by COBRA at each site, and circles indicate mean values of DNA methylation. The differences of DNA methylation are statistically significant in the gag , pol and env regions by the Mann-Whitney's U-test. Horizontal bars represent median of DNA methylation in each group. B. The relation between tax gene transcription and DNA methylation of 5'-LTR in the fresh ATL cells has been shown. DNA methylation of 5'-LTR was quantified by COBRA assay and the tax gene transcripts were detected by RT-PCR.
Figure Legend Snippet: Comparison of the DNA methylation in carriers and ATL cases. A. DNA methylation at eight different regions in the HTLV-I provirus was compared between carriers (C) and ATL cases (A). DNA methylation was quantified by COBRA in 20 carriers and 20 ATL cases. Each sample was analyzed three times by COBRA at each site, and circles indicate mean values of DNA methylation. The differences of DNA methylation are statistically significant in the gag , pol and env regions by the Mann-Whitney's U-test. Horizontal bars represent median of DNA methylation in each group. B. The relation between tax gene transcription and DNA methylation of 5'-LTR in the fresh ATL cells has been shown. DNA methylation of 5'-LTR was quantified by COBRA assay and the tax gene transcripts were detected by RT-PCR.

Techniques Used: DNA Methylation Assay, Combined Bisulfite Restriction Analysis Assay, MANN-WHITNEY, Reverse Transcription Polymerase Chain Reaction

DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.
Figure Legend Snippet: DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.

Techniques Used: DNA Methylation Assay, Methylation Sequencing, Combined Bisulfite Restriction Analysis Assay, Expressing, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Methylation

27) Product Images from "Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms"

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms

Journal: Retrovirology

doi: 10.1186/1742-4690-2-64

DNA methylation of provirus is not associated with methylated CpG sites in the genome. Integration sites of HTLV-I provirus in leukemic cells have been determined by inverse PCR, and then DNA methylation in genome has been analyzed by sodium bisulfite sequencing. DNA methylation of 5'-LTR was also analyzed by sodium bisulfite sequencing method. Vertical bars represent CpG sites. Open circle indicates unmethylated CpG site, and closed one means methylated CpG site. N: normal PBMCs from non-carrier donor.
Figure Legend Snippet: DNA methylation of provirus is not associated with methylated CpG sites in the genome. Integration sites of HTLV-I provirus in leukemic cells have been determined by inverse PCR, and then DNA methylation in genome has been analyzed by sodium bisulfite sequencing. DNA methylation of 5'-LTR was also analyzed by sodium bisulfite sequencing method. Vertical bars represent CpG sites. Open circle indicates unmethylated CpG site, and closed one means methylated CpG site. N: normal PBMCs from non-carrier donor.

Techniques Used: DNA Methylation Assay, Methylation, Inverse PCR, Methylation Sequencing

DNA methylation and histone modifications in fresh ATL cases. A. The relationships among DNA methylation, tax gene expression and histone modification in 5'-LTR were analyzed in three ATL cases. Cases 1 and 3 have one copy of the complete HTLV-I provirus, while Case 2 has a defective provirus that lacks part of the pol gene. DNA methylation was analyzed by COBRA. The tax gene transcripts could be detected in Case 1, but not in Cases 2 or 3, by RT-PCR. ChIP assays were also performed using primers for 5'-LTR to analyze acetylation of histone H3 (Ac-H3) and H4 (Ac-H4). W.C.E.: whole cell extract. B. Recovery of tax gene expression ex vivo . The PBMCs isolated from Case 3 were immediately cultured ex vivo for several hours and tested the transcription of tax mRNA by RT-PCR.
Figure Legend Snippet: DNA methylation and histone modifications in fresh ATL cases. A. The relationships among DNA methylation, tax gene expression and histone modification in 5'-LTR were analyzed in three ATL cases. Cases 1 and 3 have one copy of the complete HTLV-I provirus, while Case 2 has a defective provirus that lacks part of the pol gene. DNA methylation was analyzed by COBRA. The tax gene transcripts could be detected in Case 1, but not in Cases 2 or 3, by RT-PCR. ChIP assays were also performed using primers for 5'-LTR to analyze acetylation of histone H3 (Ac-H3) and H4 (Ac-H4). W.C.E.: whole cell extract. B. Recovery of tax gene expression ex vivo . The PBMCs isolated from Case 3 were immediately cultured ex vivo for several hours and tested the transcription of tax mRNA by RT-PCR.

Techniques Used: DNA Methylation Assay, Expressing, Modification, Combined Bisulfite Restriction Analysis Assay, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Ex Vivo, Isolation, Cell Culture

DNA methylation in ATL cell lines, HTLV-I carriers and ATL cases. The tax gene transcription in ATL cell lines was studied by RT-PCR (A), and the expression of GAPDH gene has been used as a control. DNA methylation throughout the HTLV-I provirus was studied by COBRA in tax gene-expressing (B) and non-expressing cell lines (C). Furthermore, DNA methylation was also analyzed in 20 carriers and 20 ATL cases by COBRA, and representative patterns of DNA methylation are shown in D. The number of HTLV-I provirus has been analyzed by Southern blot method, and shown in the parenthesis (B, C and D). Each bar indicates the extent of DNA methylation that was calculated by COBRA.
Figure Legend Snippet: DNA methylation in ATL cell lines, HTLV-I carriers and ATL cases. The tax gene transcription in ATL cell lines was studied by RT-PCR (A), and the expression of GAPDH gene has been used as a control. DNA methylation throughout the HTLV-I provirus was studied by COBRA in tax gene-expressing (B) and non-expressing cell lines (C). Furthermore, DNA methylation was also analyzed in 20 carriers and 20 ATL cases by COBRA, and representative patterns of DNA methylation are shown in D. The number of HTLV-I provirus has been analyzed by Southern blot method, and shown in the parenthesis (B, C and D). Each bar indicates the extent of DNA methylation that was calculated by COBRA.

Techniques Used: DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Combined Bisulfite Restriction Analysis Assay, Southern Blot

Comparison of the DNA methylation in carriers and ATL cases. A. DNA methylation at eight different regions in the HTLV-I provirus was compared between carriers (C) and ATL cases (A). DNA methylation was quantified by COBRA in 20 carriers and 20 ATL cases. Each sample was analyzed three times by COBRA at each site, and circles indicate mean values of DNA methylation. The differences of DNA methylation are statistically significant in the gag , pol and env regions by the Mann-Whitney's U-test. Horizontal bars represent median of DNA methylation in each group. B. The relation between tax gene transcription and DNA methylation of 5'-LTR in the fresh ATL cells has been shown. DNA methylation of 5'-LTR was quantified by COBRA assay and the tax gene transcripts were detected by RT-PCR.
Figure Legend Snippet: Comparison of the DNA methylation in carriers and ATL cases. A. DNA methylation at eight different regions in the HTLV-I provirus was compared between carriers (C) and ATL cases (A). DNA methylation was quantified by COBRA in 20 carriers and 20 ATL cases. Each sample was analyzed three times by COBRA at each site, and circles indicate mean values of DNA methylation. The differences of DNA methylation are statistically significant in the gag , pol and env regions by the Mann-Whitney's U-test. Horizontal bars represent median of DNA methylation in each group. B. The relation between tax gene transcription and DNA methylation of 5'-LTR in the fresh ATL cells has been shown. DNA methylation of 5'-LTR was quantified by COBRA assay and the tax gene transcripts were detected by RT-PCR.

Techniques Used: DNA Methylation Assay, Combined Bisulfite Restriction Analysis Assay, MANN-WHITNEY, Reverse Transcription Polymerase Chain Reaction

DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.
Figure Legend Snippet: DNA methylation of the HTLV-I provirus assessed by sodium bisulfite sequencing and COBRA. A. DNA methylation in the HTLV-I provirus was analyzed by sodium bisulfite sequencing in a case of acute ATL and a tax gene-expressing cell line, ATL-48T. Eight DNA regions, which were represented as bars in A, were amplified with sodium bisulfite treated DNA. The PCR products were subcloned into plasmid DNA, and then the sequences of each clone were determined for at least ten clones of each region. Arrowheads indicate the CpG sites that were target sites for COBRA. Closed circle indicates methylated CpG, and open circle means unmethylated CpG. The number of integrated provirus has been shown in parenthesis. B. Representative data of COBRA has been shown. PCR products, which were amplified with sodium bisulfite treated DNAs, were digested with Taq I or Acc II. The extent of methylation in each CpG site was measured as described in Methods, and presented as percentages of methylated CpG. The number in parenthesis represents the position of cytidine residue in analyzed CpG site by COBRA according to Seiki et al. [41]. C. DNA methylation studied by COBRA at eight points in the provirus as shown by arrowheads. Each bar represented the extent of DNA methylation at the points shown by arrowhead. The analyses by COBRA were performed three times independently, and the extents of DNA methylation are shown by the mean ± SD. The number in parenthesis shows the position of cytidine residue of CpG site analyzed by COBRA.

Techniques Used: DNA Methylation Assay, Methylation Sequencing, Combined Bisulfite Restriction Analysis Assay, Expressing, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Methylation

28) Product Images from "Galectin-1 Is Implicated in the Protein Kinase C ?/Vimentin-Controlled Trafficking of Integrin-?1 in Glioblastoma Cells"

Article Title: Galectin-1 Is Implicated in the Protein Kinase C ?/Vimentin-Controlled Trafficking of Integrin-?1 in Glioblastoma Cells

Journal: Brain Pathology (Zurich, Switzerland)

doi: 10.1111/j.1750-3639.2008.00227.x

Integrin-α9 versus integrin-β1 expression . A. Integrin-α9, integrin-β1 and actin expression across eight glioma cell lines by reverse transcription-polymerase chain reaction (RT-PCR) analysis. B. Quantitative RT-PCR analysis of integrin-α9 versus integrin-β1 expression in glioma cell lines Hs683, U87 and U373. C. Hs683 expression levels of integrin-α9 and integrin-β1 under control (ctrl), scramble-transfected (scr) or galectin-1 siRNA-transfected (siGal1) conditions obtained through microarray analysis using the Affymetrix Human Genome U133 set Plus 2.0.
Figure Legend Snippet: Integrin-α9 versus integrin-β1 expression . A. Integrin-α9, integrin-β1 and actin expression across eight glioma cell lines by reverse transcription-polymerase chain reaction (RT-PCR) analysis. B. Quantitative RT-PCR analysis of integrin-α9 versus integrin-β1 expression in glioma cell lines Hs683, U87 and U373. C. Hs683 expression levels of integrin-α9 and integrin-β1 under control (ctrl), scramble-transfected (scr) or galectin-1 siRNA-transfected (siGal1) conditions obtained through microarray analysis using the Affymetrix Human Genome U133 set Plus 2.0.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Microarray

29) Product Images from "piRNA-mediated regulation of transposon alternative splicing in soma and germline"

Article Title: piRNA-mediated regulation of transposon alternative splicing in soma and germline

Journal: Nature

doi: 10.1038/nature25018

‘ P -cytotype’ as well as piRNA pathway components involved in piRNA biogenesis and targeting regulate the splicing of the IVS3 transgenic reporter in vivo a. Ethidium bromide-stained gel displaying RT-PCR reactions with primers flanking the transgenic reporter IVS3 intron. Analysis was performed with adult ovaries of non-dysgenics and dysgenic flies grown at 18°C, or with adult ovaries of heterozygous and mutants for the piRNA components aub , vas , and Panx/Silencio . Mutant analyses were performed in a Harwich background, at 29°C. Size scale in base pairs (bp) is presented for each gel. Control reactions omitting Reverse Transcriptse (RT-) are also presented. Diagram of IVS3 transgenic reporter (as in Figure 3a) and primers (arrows) used in RT-PCR reactions are depicted in the top of the ethidium bromide-stained gel. Experiments were repeated two or more times with similar results. For gel source data, see Supplementary Figure 1 . b . RT-qPCR analysis using adult ovaries of [F1] progeny carrying the IVS3 reporter, probing spliced (IVS3 splicing, quantified using primers that specifically anneal to spliced transgenic transcripts) and total ( LacZ , quantified using primers that anneal within the LacZ coding sequence) IVS3 reporter transgenic mRNA levels. [F1] progeny was originated either from reciprocal crosses between Harwich strain and w 1118 flies, or in aub /+ heterozygous, aub mutant, vas /+ heterozygous, vas mutant, panx /+ heterozygous, and panx mutant. Results are presented as mean-fold changes in the mutants (or in dysgenic) in relation to the respective heterozygous siblings (or non-dysgenic) ± standard deviation (n > =2 independent biological replicate experiments).
Figure Legend Snippet: ‘ P -cytotype’ as well as piRNA pathway components involved in piRNA biogenesis and targeting regulate the splicing of the IVS3 transgenic reporter in vivo a. Ethidium bromide-stained gel displaying RT-PCR reactions with primers flanking the transgenic reporter IVS3 intron. Analysis was performed with adult ovaries of non-dysgenics and dysgenic flies grown at 18°C, or with adult ovaries of heterozygous and mutants for the piRNA components aub , vas , and Panx/Silencio . Mutant analyses were performed in a Harwich background, at 29°C. Size scale in base pairs (bp) is presented for each gel. Control reactions omitting Reverse Transcriptse (RT-) are also presented. Diagram of IVS3 transgenic reporter (as in Figure 3a) and primers (arrows) used in RT-PCR reactions are depicted in the top of the ethidium bromide-stained gel. Experiments were repeated two or more times with similar results. For gel source data, see Supplementary Figure 1 . b . RT-qPCR analysis using adult ovaries of [F1] progeny carrying the IVS3 reporter, probing spliced (IVS3 splicing, quantified using primers that specifically anneal to spliced transgenic transcripts) and total ( LacZ , quantified using primers that anneal within the LacZ coding sequence) IVS3 reporter transgenic mRNA levels. [F1] progeny was originated either from reciprocal crosses between Harwich strain and w 1118 flies, or in aub /+ heterozygous, aub mutant, vas /+ heterozygous, vas mutant, panx /+ heterozygous, and panx mutant. Results are presented as mean-fold changes in the mutants (or in dysgenic) in relation to the respective heterozygous siblings (or non-dysgenic) ± standard deviation (n > =2 independent biological replicate experiments).

Techniques Used: Transgenic Assay, In Vivo, Staining, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Quantitative RT-PCR, Sequencing, Standard Deviation

Analysis of P -element expression and splicing in adult ovaries of non-dysgenic and dysgenic progeny grown at 18°C a , Scatterplot showing the expression of genes (gray dots) and transposons (blue dots), as measured by RNA-seq analysis (expressed in fragments per kilobase per million fragments, FPKM, log 10 ), in adult ovaries of non-dysgenic vs. dysgenic progeny grown at 18°C. P -element expression is shown in green. Genes containing P-element insertion in Harwich strain are depicted in purple. b-c , Percentage of splicing for P -element IVS1, IVS2 ( b ), and IVS3 ( c ) splicing junctions as determined by RNA-seq analysis in non-dysgenic (green) and dysgenic (red) adult ovaries. Bars represent percentage of splicing, calculated as the number of split-reads for each splicing junction normalized to the total number of reads mapping to the same junction. Results are represented as means ± standard deviation (n=2 independent biological replicate experiments). d , Ethidium bromide-stained gel displaying RT-PCR reactions with primers flanking the P -element IVS3 intron in adult ovaries of non-dysgenic and dysgenic progeny grown at 18°C, as well as aub /+ heterozygous and aub mutant grown at 29°C. Size scale in base pairs (bp) is presented for each gel. As shown, experiments were repeated three times with similar results. For gel source data, see Supplementary Figure 1 . e , RT-qPCR analysis testing accumulation of IVS3 spliced mRNA on non-dysgenic and dysgenic progeny (ovaries) grown at 18°C. Results are expressed as means of percentage of expression relative to controls ± standard deviation (n=3 independent biological replicate experiments). f, Genome-wide analysis of splicing changes in in adult ovaries of non-dysgenic vs. dysgenic progeny grown at 18°C. Quantification of splicing changes was performed using RNA-seq data and the JUM method 44,45 . Results are expressed as log 2 fold changes in splicing (dysgenic/non-dysgenic). Gray dots represent individual splice junctions identified, sorted by fold change values. Green dots represent splice junctions with statistically significant changes in heterozygous vs. mutant comparisons (adjust p -value
Figure Legend Snippet: Analysis of P -element expression and splicing in adult ovaries of non-dysgenic and dysgenic progeny grown at 18°C a , Scatterplot showing the expression of genes (gray dots) and transposons (blue dots), as measured by RNA-seq analysis (expressed in fragments per kilobase per million fragments, FPKM, log 10 ), in adult ovaries of non-dysgenic vs. dysgenic progeny grown at 18°C. P -element expression is shown in green. Genes containing P-element insertion in Harwich strain are depicted in purple. b-c , Percentage of splicing for P -element IVS1, IVS2 ( b ), and IVS3 ( c ) splicing junctions as determined by RNA-seq analysis in non-dysgenic (green) and dysgenic (red) adult ovaries. Bars represent percentage of splicing, calculated as the number of split-reads for each splicing junction normalized to the total number of reads mapping to the same junction. Results are represented as means ± standard deviation (n=2 independent biological replicate experiments). d , Ethidium bromide-stained gel displaying RT-PCR reactions with primers flanking the P -element IVS3 intron in adult ovaries of non-dysgenic and dysgenic progeny grown at 18°C, as well as aub /+ heterozygous and aub mutant grown at 29°C. Size scale in base pairs (bp) is presented for each gel. As shown, experiments were repeated three times with similar results. For gel source data, see Supplementary Figure 1 . e , RT-qPCR analysis testing accumulation of IVS3 spliced mRNA on non-dysgenic and dysgenic progeny (ovaries) grown at 18°C. Results are expressed as means of percentage of expression relative to controls ± standard deviation (n=3 independent biological replicate experiments). f, Genome-wide analysis of splicing changes in in adult ovaries of non-dysgenic vs. dysgenic progeny grown at 18°C. Quantification of splicing changes was performed using RNA-seq data and the JUM method 44,45 . Results are expressed as log 2 fold changes in splicing (dysgenic/non-dysgenic). Gray dots represent individual splice junctions identified, sorted by fold change values. Green dots represent splice junctions with statistically significant changes in heterozygous vs. mutant comparisons (adjust p -value

Techniques Used: Expressing, RNA Sequencing Assay, Standard Deviation, Staining, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Quantitative RT-PCR, Genome Wide

piRNAs, but not siRNAs, modulate P -element splicing in germ cells All analyses were performed in a Harwich background. a , RT-qPCR analysis on piRNA- ( piwi , aub , ago3 , spnE , and vas ) and siRNA-biogenesis ( dcr2 and ago2 ) mutant adult ovaries. Results are presented as means of fold changes in mutants in relation to respective heterozygote ± standard deviation (n > =2 independent biological replicate experiments). b , Ethidium bromide-stained gel displaying RT-PCR reactions with primers flanking the P -element IVS3 intron in piRNA- and siRNA-biogenesis mutants. Size scale in base pairs (bp) is presented for each gel. Experiments were repeated two times with similar results. For gel source data, see Supplementary Figure 1 . c , Density plots for normalized strand-specific mRNA steady-state levels (measured by RNA-seq and represented as reads per million, RPM) over consensus P -element sequence (top diagram) in aub /+ heterozygous (yellow, top plot) and aub mutant (blue, bottom plot) adult ovaries. The number and position of split-reads (represented by arcs that connect exons) observed for IVS1, IVS2, and IVS3 splicing junctions is shown below each density plot. Experiments were repeated two times with similar results. d-e , Percentage of splicing for P -element IVS1, IVS2 ( d ), and IVS3 ( e ). Splicing was quantified using RNA-seq analysis in aub /+ heterozygous (yellow), aub mutant (blue), piwi /+ heterozygous (beige), and piwi mutant (purple) adult ovaries. Percentage of splicing was calculated as the number of split-reads for each splicing junction normalized to the total number of reads mapping to the same junction. Results are represented as means ± standard deviation (n=2 independent biological replicate experiments). f , Representative confocal projections of RNA-FISH signal (grayscale) showing the accumulation of sense RNA for Burdock and P -element transposons in heterozygous and mutant egg chambers. Bottom panels depict projections of representative nurse cell nuclei (purple dotted line) for the same genotypes. Scale bars, 20 μM. Experiments were repeated two or more times with similar results.
Figure Legend Snippet: piRNAs, but not siRNAs, modulate P -element splicing in germ cells All analyses were performed in a Harwich background. a , RT-qPCR analysis on piRNA- ( piwi , aub , ago3 , spnE , and vas ) and siRNA-biogenesis ( dcr2 and ago2 ) mutant adult ovaries. Results are presented as means of fold changes in mutants in relation to respective heterozygote ± standard deviation (n > =2 independent biological replicate experiments). b , Ethidium bromide-stained gel displaying RT-PCR reactions with primers flanking the P -element IVS3 intron in piRNA- and siRNA-biogenesis mutants. Size scale in base pairs (bp) is presented for each gel. Experiments were repeated two times with similar results. For gel source data, see Supplementary Figure 1 . c , Density plots for normalized strand-specific mRNA steady-state levels (measured by RNA-seq and represented as reads per million, RPM) over consensus P -element sequence (top diagram) in aub /+ heterozygous (yellow, top plot) and aub mutant (blue, bottom plot) adult ovaries. The number and position of split-reads (represented by arcs that connect exons) observed for IVS1, IVS2, and IVS3 splicing junctions is shown below each density plot. Experiments were repeated two times with similar results. d-e , Percentage of splicing for P -element IVS1, IVS2 ( d ), and IVS3 ( e ). Splicing was quantified using RNA-seq analysis in aub /+ heterozygous (yellow), aub mutant (blue), piwi /+ heterozygous (beige), and piwi mutant (purple) adult ovaries. Percentage of splicing was calculated as the number of split-reads for each splicing junction normalized to the total number of reads mapping to the same junction. Results are represented as means ± standard deviation (n=2 independent biological replicate experiments). f , Representative confocal projections of RNA-FISH signal (grayscale) showing the accumulation of sense RNA for Burdock and P -element transposons in heterozygous and mutant egg chambers. Bottom panels depict projections of representative nurse cell nuclei (purple dotted line) for the same genotypes. Scale bars, 20 μM. Experiments were repeated two or more times with similar results.

Techniques Used: Quantitative RT-PCR, Mutagenesis, Standard Deviation, Staining, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Sequencing, Fluorescence In Situ Hybridization

30) Product Images from "Decreased endothelin receptor B expression in large primary uveal melanomas is associated with early clinical metastasis and short survival"

Article Title: Decreased endothelin receptor B expression in large primary uveal melanomas is associated with early clinical metastasis and short survival

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6600620

Comparative multiplex RT–PCR of EDNRB and the control gene KIAA0228 in SCLCs. N: cDNA from matched normal tissue. T: Tumour cDNA.
Figure Legend Snippet: Comparative multiplex RT–PCR of EDNRB and the control gene KIAA0228 in SCLCs. N: cDNA from matched normal tissue. T: Tumour cDNA.

Techniques Used: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction

31) Product Images from "Targeting the VEGF-C/VEGFR3 axis suppresses Slug-mediated cancer metastasis and stemness via inhibition of KRAS/YAP1 signaling"

Article Title: Targeting the VEGF-C/VEGFR3 axis suppresses Slug-mediated cancer metastasis and stemness via inhibition of KRAS/YAP1 signaling

Journal: Oncotarget

doi: 10.18632/oncotarget.13629

Anti-VEGFR3 peptide represses VEGF-C-induced signaling, cell mobility and cancer stemness in skin cancer a . A transwell assaydemonstrated the migration and invasion abilities in BCC cells pre-incubated with rhVEGF-C followed by anti-VEGFR3 (anti-R3) or control (Ctrl) peptide treatment. b . Western blot analysis of indicated proteins in rhVEGF-C-pre-incubated cells treated with anti-R3 or Ctrl peptide. c . qRT-PCR analysisof SOX2 and OCT4 mRNA expression in indicated cells. d . ALDH activity in indicated cells was analyzed by flow cytometry. e . A transwell assay showed migration and invasion abilities in indicated cells. The results are shown as the mean ± SD of three independent experiments, each performed in triplicate. * P
Figure Legend Snippet: Anti-VEGFR3 peptide represses VEGF-C-induced signaling, cell mobility and cancer stemness in skin cancer a . A transwell assaydemonstrated the migration and invasion abilities in BCC cells pre-incubated with rhVEGF-C followed by anti-VEGFR3 (anti-R3) or control (Ctrl) peptide treatment. b . Western blot analysis of indicated proteins in rhVEGF-C-pre-incubated cells treated with anti-R3 or Ctrl peptide. c . qRT-PCR analysisof SOX2 and OCT4 mRNA expression in indicated cells. d . ALDH activity in indicated cells was analyzed by flow cytometry. e . A transwell assay showed migration and invasion abilities in indicated cells. The results are shown as the mean ± SD of three independent experiments, each performed in triplicate. * P

Techniques Used: Migration, Incubation, Western Blot, Quantitative RT-PCR, Expressing, Activity Assay, Flow Cytometry, Cytometry, Transwell Assay

VEGF-C enhances the cancer stemness properties in skin cancer a-g . QRT-PCR analysis of the SOX2 , OCT4 , KLF4 , NANOG , CD133 , CD34 and CD44 mRNA expression in indicated cells with rhVEGF-C (200 ng/ml) treatment. h . ALDH activity of the indicated cells was measured by flow cytometry. i-q . Expression of VEGF-C (i) and Slug (j) was detected, and Slug expression restores the expression of CSC markers ( SOX2 (k), OCT4 (l), KLF4 (m), NANOG (n), CD133 (o), CD34 (p) and CD44 (q)) in indicated cells by QRT-PCR analysis. r . Slug expression recovers ALDH activity in indicated cells by flow cytometry.
Figure Legend Snippet: VEGF-C enhances the cancer stemness properties in skin cancer a-g . QRT-PCR analysis of the SOX2 , OCT4 , KLF4 , NANOG , CD133 , CD34 and CD44 mRNA expression in indicated cells with rhVEGF-C (200 ng/ml) treatment. h . ALDH activity of the indicated cells was measured by flow cytometry. i-q . Expression of VEGF-C (i) and Slug (j) was detected, and Slug expression restores the expression of CSC markers ( SOX2 (k), OCT4 (l), KLF4 (m), NANOG (n), CD133 (o), CD34 (p) and CD44 (q)) in indicated cells by QRT-PCR analysis. r . Slug expression recovers ALDH activity in indicated cells by flow cytometry.

Techniques Used: Quantitative RT-PCR, Expressing, Activity Assay, Flow Cytometry, Cytometry

VEGF-C increases Slug expression, cell migration, invasion and stemness through YAP1 a . (Upper panel) Western blot analysis of Slug and YAP1 in indicated cells. Tubulin was used as a loading control. (Lower panel) QRT-PCR analysis of VEGF-C expression in indicated cells. b . A transwell assay demonstrated the migration and invasion abilities of indicated cells. c and d . QRT-PCR analysis of SOX2 and OCT4 mRNA expression in indicated cells. e . ALDH activity of indicated cells was determined by flow cytometry. f-h . The Oncomine database was used to analyze the correlation of VEGF-C, YAP1 and Slug with survival rate in skin cancer patients. The results are shown as the mean ± SD of three independent experiments, each performed in triplicate. * P
Figure Legend Snippet: VEGF-C increases Slug expression, cell migration, invasion and stemness through YAP1 a . (Upper panel) Western blot analysis of Slug and YAP1 in indicated cells. Tubulin was used as a loading control. (Lower panel) QRT-PCR analysis of VEGF-C expression in indicated cells. b . A transwell assay demonstrated the migration and invasion abilities of indicated cells. c and d . QRT-PCR analysis of SOX2 and OCT4 mRNA expression in indicated cells. e . ALDH activity of indicated cells was determined by flow cytometry. f-h . The Oncomine database was used to analyze the correlation of VEGF-C, YAP1 and Slug with survival rate in skin cancer patients. The results are shown as the mean ± SD of three independent experiments, each performed in triplicate. * P

Techniques Used: Expressing, Migration, Western Blot, Quantitative RT-PCR, Transwell Assay, Activity Assay, Flow Cytometry, Cytometry

VEGF-C increases the migration and invasion abilities of skin cancer cells a . BCC and A2058 cells were incubated with 200 ng/ml of VEGF-C for the indicated time points, and phosphorylated VEGFR3 and VEGFR3 were detected by Western blot analysis. Tubulin was used as a loading control. b . The mRNA expression of VEGF-C was analyzed by real-time qRT-PCR. c and d . A transwell assay was performed to examine cell migration and invasion abilities. Cell motility and phosphorylation of VEGFR3 was inhibited by knockdown of VEGF-C and recovered by treatment with rhVEGF-C by transwell assay and Western blot analysis, respectively. e . Oncomine database analysis showed elevated VEGF-C expression in metastatic melanoma tissues. f . Expression of EMT-inducing transcription factors (Slug, ZEB1, Twist and Snail) was determined by Western blot analysis. g . The effects of Slug expression on skin cancer cell motility were determined by transwell assay. Western blot analysis was used to confirm the expression of the indicated proteins. The results are shown as the mean ± SD of three independent experiments, each performed in triplicate. * P
Figure Legend Snippet: VEGF-C increases the migration and invasion abilities of skin cancer cells a . BCC and A2058 cells were incubated with 200 ng/ml of VEGF-C for the indicated time points, and phosphorylated VEGFR3 and VEGFR3 were detected by Western blot analysis. Tubulin was used as a loading control. b . The mRNA expression of VEGF-C was analyzed by real-time qRT-PCR. c and d . A transwell assay was performed to examine cell migration and invasion abilities. Cell motility and phosphorylation of VEGFR3 was inhibited by knockdown of VEGF-C and recovered by treatment with rhVEGF-C by transwell assay and Western blot analysis, respectively. e . Oncomine database analysis showed elevated VEGF-C expression in metastatic melanoma tissues. f . Expression of EMT-inducing transcription factors (Slug, ZEB1, Twist and Snail) was determined by Western blot analysis. g . The effects of Slug expression on skin cancer cell motility were determined by transwell assay. Western blot analysis was used to confirm the expression of the indicated proteins. The results are shown as the mean ± SD of three independent experiments, each performed in triplicate. * P

Techniques Used: Migration, Incubation, Western Blot, Expressing, Quantitative RT-PCR, Transwell Assay

32) Product Images from "Transcriptional Profiling of Chondrodysplasia Growth Plate Cartilage Reveals Adaptive ER-Stress Networks That Allow Survival but Disrupt Hypertrophy"

Article Title: Transcriptional Profiling of Chondrodysplasia Growth Plate Cartilage Reveals Adaptive ER-Stress Networks That Allow Survival but Disrupt Hypertrophy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0024600

Expression of wildtype hypertrophic and proliferative growth plate zone gene signatures in Schmid and Cog hypertrophic zones. (A) Heatmap depicting the relative fold difference (log fold change) of 510 wildtype (Wt) hypertrophic zone (HZ) signature genes following the comparison of datasets generated by microarray analyses of Wt proliferative zone (PZ), Wt HZ, homozygous Schmid (Schmid) HZ, or Tg cog (Cog) HZ aRNA (N = 3). (B) Heatmap depicting the relative expression (log fold change) of 773 Wt PZ signature genes following the comparison of datasets generated by microarray analyses of Wt PZ, Wt HZ, Schmid HZ, or Cog HZ aRNA (N = 3). For both heatmaps, each Wt growth plate zone signature gene is represented by a single bar, colour-coded according to relative expression as indicated, with downregulated genes coloured yellow, and upregulated genes coloured red. (C–F) Validation of (A) and (B) by quantitative PCR (qPCR) using (C) Wt PZ and Wt HZ cDNA for markers of the Wt HZ gene signature, (D) Wt PZ and Wt HZ cDNA for markers of the Wt PZ gene signature, (E) Schmid HZ and Wt HZ cDNA for the Wt HZ markers used in (C), and (F) Schmid HZ and Wt HZ cDNA for the Wt PZ markers used in (D); N = 3, expression profiles expressed as Fold Difference versus Wt HZ, microarray data shaded dark grey, qPCR data shaded light grey, qPCR error bars indicate standard deviation around the mean.
Figure Legend Snippet: Expression of wildtype hypertrophic and proliferative growth plate zone gene signatures in Schmid and Cog hypertrophic zones. (A) Heatmap depicting the relative fold difference (log fold change) of 510 wildtype (Wt) hypertrophic zone (HZ) signature genes following the comparison of datasets generated by microarray analyses of Wt proliferative zone (PZ), Wt HZ, homozygous Schmid (Schmid) HZ, or Tg cog (Cog) HZ aRNA (N = 3). (B) Heatmap depicting the relative expression (log fold change) of 773 Wt PZ signature genes following the comparison of datasets generated by microarray analyses of Wt PZ, Wt HZ, Schmid HZ, or Cog HZ aRNA (N = 3). For both heatmaps, each Wt growth plate zone signature gene is represented by a single bar, colour-coded according to relative expression as indicated, with downregulated genes coloured yellow, and upregulated genes coloured red. (C–F) Validation of (A) and (B) by quantitative PCR (qPCR) using (C) Wt PZ and Wt HZ cDNA for markers of the Wt HZ gene signature, (D) Wt PZ and Wt HZ cDNA for markers of the Wt PZ gene signature, (E) Schmid HZ and Wt HZ cDNA for the Wt HZ markers used in (C), and (F) Schmid HZ and Wt HZ cDNA for the Wt PZ markers used in (D); N = 3, expression profiles expressed as Fold Difference versus Wt HZ, microarray data shaded dark grey, qPCR data shaded light grey, qPCR error bars indicate standard deviation around the mean.

Techniques Used: Expressing, Generated, Microarray, Real-time Polymerase Chain Reaction, Standard Deviation

33) Product Images from "c-Crk proto-oncogene contributes to transcriptional repression of p120-catenin in non-small cell lung cancer cells"

Article Title: c-Crk proto-oncogene contributes to transcriptional repression of p120-catenin in non-small cell lung cancer cells

Journal: Clinical & Experimental Metastasis

doi: 10.1007/s10585-011-9378-8

a Western blots showing the expression levels of CRK-I/II in a panel of NSCLC cells. b Conventional RT-PCR measuring mRNA expression level of CRK-I/II in these NSCLC cells. Cell lines with lower p120ctn show higher CRK-I/II mRNA and protein levels
Figure Legend Snippet: a Western blots showing the expression levels of CRK-I/II in a panel of NSCLC cells. b Conventional RT-PCR measuring mRNA expression level of CRK-I/II in these NSCLC cells. Cell lines with lower p120ctn show higher CRK-I/II mRNA and protein levels

Techniques Used: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

a p120ctn mRNA levels measured by qRT-PCR following silencing CRK in A549, H157 and H358 cells. b p120ctn promoter activity in the above mentioned cells following silencing CRK. c qRT-PCR showing the efficiency of CRK silencing in these cell lines. (2 tailed student’s t -test: * P
Figure Legend Snippet: a p120ctn mRNA levels measured by qRT-PCR following silencing CRK in A549, H157 and H358 cells. b p120ctn promoter activity in the above mentioned cells following silencing CRK. c qRT-PCR showing the efficiency of CRK silencing in these cell lines. (2 tailed student’s t -test: * P

Techniques Used: Quantitative RT-PCR, Activity Assay

a Promoter analysis of p120ctn in A549 (NSCLC) and BEAS-2B (immortalized normal human epithelium) cell lines. Results are normalized for transient transfection efficiency (i.e. firefly luciferase activity) by co-transfection of a Renilla luciferase expressing control vector (pRL-SV40). Numbers on horizontal axis indicate quantity of DNA transfected. Promoter activity of A549 cells are significantly reduced compared to BEAS-2B cells. b Analysis of p120ctn promoter by creating serial deletion constructs. These deletion constructs of full length p120ctn promoter were prepared by deletions from both 5′ and 3′ ends of the p120ctn short -luc construct. Both A549 and BEAS-2B cells were transfected with this mutant constructs and 24 h later cells were lysed and subjected to dual Luciferase assay, data only shown for A549 cells. Relative promoter activity is normalized to full length promoter activity in A549 cells. p120ctn promoter activity significantly changes upon deletion segments (−9 to +36) and (+267 to +320). c Chromatin immunoprecipitation (ChIP) in A549 and H157 cells; immunoprecipitation with anti-SP1 antibody or IgG control, PCR with primers encompassing segment (−56 to +111) of the p 120ctn promoter. Error bars represent 95% confidence interval.SP1 binds to the p120ctn promoter. d Schematic view of the p120ctn core promoter region and putative cis -acting elements both at the 5′ and 3′ sides of transcription initiation site that were predicted by Alibaba2 analysis. Binding sites for transcription factors NF-1, AP-2 and SP1 are predicted within segment (−9 to +36) of the p120ctn promoter
Figure Legend Snippet: a Promoter analysis of p120ctn in A549 (NSCLC) and BEAS-2B (immortalized normal human epithelium) cell lines. Results are normalized for transient transfection efficiency (i.e. firefly luciferase activity) by co-transfection of a Renilla luciferase expressing control vector (pRL-SV40). Numbers on horizontal axis indicate quantity of DNA transfected. Promoter activity of A549 cells are significantly reduced compared to BEAS-2B cells. b Analysis of p120ctn promoter by creating serial deletion constructs. These deletion constructs of full length p120ctn promoter were prepared by deletions from both 5′ and 3′ ends of the p120ctn short -luc construct. Both A549 and BEAS-2B cells were transfected with this mutant constructs and 24 h later cells were lysed and subjected to dual Luciferase assay, data only shown for A549 cells. Relative promoter activity is normalized to full length promoter activity in A549 cells. p120ctn promoter activity significantly changes upon deletion segments (−9 to +36) and (+267 to +320). c Chromatin immunoprecipitation (ChIP) in A549 and H157 cells; immunoprecipitation with anti-SP1 antibody or IgG control, PCR with primers encompassing segment (−56 to +111) of the p 120ctn promoter. Error bars represent 95% confidence interval.SP1 binds to the p120ctn promoter. d Schematic view of the p120ctn core promoter region and putative cis -acting elements both at the 5′ and 3′ sides of transcription initiation site that were predicted by Alibaba2 analysis. Binding sites for transcription factors NF-1, AP-2 and SP1 are predicted within segment (−9 to +36) of the p120ctn promoter

Techniques Used: Transfection, Luciferase, Activity Assay, Cotransfection, Expressing, Plasmid Preparation, Construct, Mutagenesis, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Binding Assay

a Western blots showing the expression level of E-cadherin, p120ctn and SP1 in a panel of NSCLC and BEAS-2B cells. b Conventional RT-PCR analysis of mRNA for candidate SP1 binary interaction partners including CRK, MYC, E2F and SRC in the above mentioned panel of cells. c Quantitative RT-PCR measuring CRK mRNA levels in our panel of NSCLC as well as BEAS-2B cells. CRK mRNA levels inversely correlates with p120ctn levels in NSCLC cells
Figure Legend Snippet: a Western blots showing the expression level of E-cadherin, p120ctn and SP1 in a panel of NSCLC and BEAS-2B cells. b Conventional RT-PCR analysis of mRNA for candidate SP1 binary interaction partners including CRK, MYC, E2F and SRC in the above mentioned panel of cells. c Quantitative RT-PCR measuring CRK mRNA levels in our panel of NSCLC as well as BEAS-2B cells. CRK mRNA levels inversely correlates with p120ctn levels in NSCLC cells

Techniques Used: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

34) Product Images from "A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus"

Article Title: A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

Journal: BMC Genomics

doi: 10.1186/1471-2164-10-428

Expression analysis of transgenic Arabidopsis plants that overexpress the CitrSEP gene . A , Semi-quantitative RT-PCR analysis of 12-day old untransformed Col-0 and transgenic lines L120-5 and L120-9. B , qRT-PCR of CitrSEP and endogenous SEP3 in transgenic lines. Expression was normalized to the expression of the constitutive EF-1-α gene, and then to the expression in Col-0 plants. For normalization purposes, the detection level of our qRT-PCR analysis was used as an estimate of CitrSEP expression in Col-0. Expression level is indicated in the plot. nd, not detected.
Figure Legend Snippet: Expression analysis of transgenic Arabidopsis plants that overexpress the CitrSEP gene . A , Semi-quantitative RT-PCR analysis of 12-day old untransformed Col-0 and transgenic lines L120-5 and L120-9. B , qRT-PCR of CitrSEP and endogenous SEP3 in transgenic lines. Expression was normalized to the expression of the constitutive EF-1-α gene, and then to the expression in Col-0 plants. For normalization purposes, the detection level of our qRT-PCR analysis was used as an estimate of CitrSEP expression in Col-0. Expression level is indicated in the plot. nd, not detected.

Techniques Used: Expressing, Transgenic Assay, Quantitative RT-PCR

35) Product Images from "Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates"

Article Title: Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

Journal: BMC Evolutionary Biology

doi: 10.1186/1471-2148-7-113

Analysis of the cloned zebrafish β1 subunit gene and novel splice variants . A) Alignment of cloned human, rat, and zebrafish β1 amino acid sequences. Black = identical in all three species; grey = identical in 2/3 species or conserved substitution. Shown for zebrafish is the most conserved β1 splice form (variant D). Hs = Homo sapiens , Rn = Rattus norvegicus , z = zebrafish; DS* = cysteine residue predicted to participate in a disulfide bridge, based on the myelin P0 protein crystal structure; DS = predicted second disulfide bridge; N = predicted N-linked glycosylation site (N1 = human/rat, N2 = zebrafish); M1 = site of epilepsy-causing deletion (I70_E74del) in Hsβ1; M2 = site of second epilepsy-causing mutation (C121W) in Hsβ1; M3/4 = site of third and fourth epilepsy-causing mutations (R85C, R85H); S1 = nonsynonymous Hsβ1 single nucleotide polymorphism (SNP, G/A > R85H); S2 = nonsynonymous Hsβ1 SNP (C/T > T189M); P = phosphorylation site (tyrosine Y181) that regulates ankyrin recruitment (NOTE: Y200 = Y181 following cleavage of 19 amino acid signal peptide); IN = putative internalization sequence. Consensus sequence for V-type IG domain is depicted beneath the alignment: G = glycine, x = any residue, ^ = hydrophobic residues, C = cysteine, - = gap in alignment with consensus sequence, W = tryptophan, * = basic residue, L = leucine, D = aspartic acid, = glycine, alanine, or aspartate, and Y = tyrosine. Red indicates zebrafish residues that deviate from the consensus sequence. See Results for references supporting sequence annotation. B) 5' and 3' RLM-RACE PCR and RT-PCR identified four distinct splice variants expressed from zβ1 locus on zebrafish chromosome 16 ( Ensembl ). C ) Splice donor and acceptor sites of zebrafish β1 splice variants, derived from comparing cloned cDNA against genomic DNA sequences ( Ensembl ). Consensus GT-AG splice sites are labeled in red. A splice-site deviating from the consensus appears in grey. D ) Schematic diagram of β1 splice variants A, B, and D, whose predicted proteins differ only in the length of their intracytoplasmic C-terminal tail. S-S = disulfide bridge. NH 3 = 5' amino terminus, CO 2 = 3' carboxyl terminus, β = putative N-linked glyosylation site. Alignment of C-terminal tail of variants zβ1A, zβ1B, and zβ1D (below). zβ1C is not shown as it is predicted to lack both extracellular IG and transmembrane domains.
Figure Legend Snippet: Analysis of the cloned zebrafish β1 subunit gene and novel splice variants . A) Alignment of cloned human, rat, and zebrafish β1 amino acid sequences. Black = identical in all three species; grey = identical in 2/3 species or conserved substitution. Shown for zebrafish is the most conserved β1 splice form (variant D). Hs = Homo sapiens , Rn = Rattus norvegicus , z = zebrafish; DS* = cysteine residue predicted to participate in a disulfide bridge, based on the myelin P0 protein crystal structure; DS = predicted second disulfide bridge; N = predicted N-linked glycosylation site (N1 = human/rat, N2 = zebrafish); M1 = site of epilepsy-causing deletion (I70_E74del) in Hsβ1; M2 = site of second epilepsy-causing mutation (C121W) in Hsβ1; M3/4 = site of third and fourth epilepsy-causing mutations (R85C, R85H); S1 = nonsynonymous Hsβ1 single nucleotide polymorphism (SNP, G/A > R85H); S2 = nonsynonymous Hsβ1 SNP (C/T > T189M); P = phosphorylation site (tyrosine Y181) that regulates ankyrin recruitment (NOTE: Y200 = Y181 following cleavage of 19 amino acid signal peptide); IN = putative internalization sequence. Consensus sequence for V-type IG domain is depicted beneath the alignment: G = glycine, x = any residue, ^ = hydrophobic residues, C = cysteine, - = gap in alignment with consensus sequence, W = tryptophan, * = basic residue, L = leucine, D = aspartic acid, = glycine, alanine, or aspartate, and Y = tyrosine. Red indicates zebrafish residues that deviate from the consensus sequence. See Results for references supporting sequence annotation. B) 5' and 3' RLM-RACE PCR and RT-PCR identified four distinct splice variants expressed from zβ1 locus on zebrafish chromosome 16 ( Ensembl ). C ) Splice donor and acceptor sites of zebrafish β1 splice variants, derived from comparing cloned cDNA against genomic DNA sequences ( Ensembl ). Consensus GT-AG splice sites are labeled in red. A splice-site deviating from the consensus appears in grey. D ) Schematic diagram of β1 splice variants A, B, and D, whose predicted proteins differ only in the length of their intracytoplasmic C-terminal tail. S-S = disulfide bridge. NH 3 = 5' amino terminus, CO 2 = 3' carboxyl terminus, β = putative N-linked glyosylation site. Alignment of C-terminal tail of variants zβ1A, zβ1B, and zβ1D (below). zβ1C is not shown as it is predicted to lack both extracellular IG and transmembrane domains.

Techniques Used: Clone Assay, Variant Assay, Mutagenesis, Sequencing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Labeling

36) Product Images from "Culture-Independent Identification of Mycobacterium avium Subspecies paratuberculosis in Ovine Tissues: Comparison with Bacterial Culture and Histopathological Lesions"

Article Title: Culture-Independent Identification of Mycobacterium avium Subspecies paratuberculosis in Ovine Tissues: Comparison with Bacterial Culture and Histopathological Lesions

Journal: Frontiers in Veterinary Science

doi: 10.3389/fvets.2017.00232

(A) Scatter plot of mean log 10 DNA quantified by mesenteric lymph node (MLN) quantitative PCR (qPCR) against log 10 DNA quantified by intestinal tissue (gut) qPCR. The solid line is the regression line. A positive correlation coefficient of 0.78 (95% CI: 0.69, 0.84) was observed. (B) Bland–Altman plot depicting difference of log 10 DNA quantified by gut qPCR and MLN qPCR against average log 10 DNA quantified by them. The dotted line shows the line of perfect agreement whereas two solid lines show the limit of agreement. The DNA quantified by gut qPCR was 2.94 times higher than that quantified by MLN qPCR for the same animal on an average. The solid line is the regression line of average log 10 DNA against difference of log 10 DNA. An increase in average difference was observed for the increase in DNA quantity in the gut and MLN homogenate.
Figure Legend Snippet: (A) Scatter plot of mean log 10 DNA quantified by mesenteric lymph node (MLN) quantitative PCR (qPCR) against log 10 DNA quantified by intestinal tissue (gut) qPCR. The solid line is the regression line. A positive correlation coefficient of 0.78 (95% CI: 0.69, 0.84) was observed. (B) Bland–Altman plot depicting difference of log 10 DNA quantified by gut qPCR and MLN qPCR against average log 10 DNA quantified by them. The dotted line shows the line of perfect agreement whereas two solid lines show the limit of agreement. The DNA quantified by gut qPCR was 2.94 times higher than that quantified by MLN qPCR for the same animal on an average. The solid line is the regression line of average log 10 DNA against difference of log 10 DNA. An increase in average difference was observed for the increase in DNA quantity in the gut and MLN homogenate.

Techniques Used: Real-time Polymerase Chain Reaction

IS 1311 PCR-restriction endonuclease analysis profiles for DNA extracted from tissues. Lanes 1, 2, and 4 are IS 1311 PCR products from DNA extracted from gut tissues restricted with Mse I and Hinf I enzymes. Lanes 3 and 5 are IS 1311 PCR products from DNA extracted from lymph node tissues restricted with Mse I and Hinf I enzymes. Lane 6 is negative control. Lane 7 is IS 1311 PCR product from S strain restricted with Mse I and Hinf I enzymes. Lane 8 is the unrestricted IS 1311 PCR product from S strain. Lane 9 is the molecular size markers (Number VIII).
Figure Legend Snippet: IS 1311 PCR-restriction endonuclease analysis profiles for DNA extracted from tissues. Lanes 1, 2, and 4 are IS 1311 PCR products from DNA extracted from gut tissues restricted with Mse I and Hinf I enzymes. Lanes 3 and 5 are IS 1311 PCR products from DNA extracted from lymph node tissues restricted with Mse I and Hinf I enzymes. Lane 6 is negative control. Lane 7 is IS 1311 PCR product from S strain restricted with Mse I and Hinf I enzymes. Lane 8 is the unrestricted IS 1311 PCR product from S strain. Lane 9 is the molecular size markers (Number VIII).

Techniques Used: Polymerase Chain Reaction, Negative Control

37) Product Images from "Candidate target genes for loss of heterozygosity on human chromosome 17q21"

Article Title: Candidate target genes for loss of heterozygosity on human chromosome 17q21

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6601848

Methylation-specific PCR of the JUP promoter region in primary breast tumour DNAs having LOH on chromosome 17q21. ( A ) Test of primer pairs for PCR of unmethylated and in vitro methylated recombinant genomic DNA containing the JUP promoter region: lane 1, Marker Hae III-digested phi X174 DNA; lane 2, methylated-specifc primers and methylated JUP genomic DNA; lane 3, methylated specifc primers and unmethylated JUP genomic DNA; lane 4, unmethylated specifc primers and methylated JUP genomic DNA; lane 5, unmethylated specifc primers and unmethylated JUP genomic DNA. PCR analysis of primary breast tumour DNAs with methylation specific primers ( B ) or primers specific for unmethylated JUP genomic DNA ( C ). The DNAs were: lane1, Marker Hae III-digested phi X174 DNA; lane 2, tumour 16; lane 3, tumour 20; lane 4, tumour 26; lane 5, tumour30; lane 6, tumour 44; lane 7, tumour 62; lane 8, tumour 63; lane 9, tumour 89; lane 10, tumour 117; lane 11, tumour127; lane 12, unrelated genomic DNA; lane 13, control methylated recombinant JUP promoter region ( B ) and control unmethylated recombinant JUP promoter region (C); lane 14, water control.
Figure Legend Snippet: Methylation-specific PCR of the JUP promoter region in primary breast tumour DNAs having LOH on chromosome 17q21. ( A ) Test of primer pairs for PCR of unmethylated and in vitro methylated recombinant genomic DNA containing the JUP promoter region: lane 1, Marker Hae III-digested phi X174 DNA; lane 2, methylated-specifc primers and methylated JUP genomic DNA; lane 3, methylated specifc primers and unmethylated JUP genomic DNA; lane 4, unmethylated specifc primers and methylated JUP genomic DNA; lane 5, unmethylated specifc primers and unmethylated JUP genomic DNA. PCR analysis of primary breast tumour DNAs with methylation specific primers ( B ) or primers specific for unmethylated JUP genomic DNA ( C ). The DNAs were: lane1, Marker Hae III-digested phi X174 DNA; lane 2, tumour 16; lane 3, tumour 20; lane 4, tumour 26; lane 5, tumour30; lane 6, tumour 44; lane 7, tumour 62; lane 8, tumour 63; lane 9, tumour 89; lane 10, tumour 117; lane 11, tumour127; lane 12, unrelated genomic DNA; lane 13, control methylated recombinant JUP promoter region ( B ) and control unmethylated recombinant JUP promoter region (C); lane 14, water control.

Techniques Used: Methylation, Polymerase Chain Reaction, In Vitro, Recombinant, Marker

( A ) A partial map of human chromosome 17q12–q21. The PCR products of an analysis of the P1-phage clones are shown for the presence of exons corresponding to the indicated genes. The arabic numbers represent the templates used to perform the PCR amplification of the indicated exon: lane 1, water control; lane 2, 122F4; lane 3, 50H1 P1-phage clones; lane 4, genomic DNA control. The intron/exon primers of all the genes are available upon request. The sizes of the PCR-amplified fragments are indicated in bp. The arrow indicates transcriptional orientation 5′–3′ of the indicated genes. The portions of the map with right-hand hatch marks correspond to regions in which the nucleotide sequence of the exon/intron junctions was determined. The regions of the map in which the entire nucleotide sequence was determined are indicated with left-hand hatch marks. The centromeric end of the map is on the left and the telomeric end is on the right side. ( B ) A map of the region of chromosome 17q12–q21 affected by LOH. The positions of Exons 1A, 1B, the remainder of JUP exons and HAP1 as well as D17S846, 122F4-3′ and D17S746 on the chromosome map are indicated. The PCR products of an analysis of the P1-phage clones, 50H1 and 122F4, are shown for the presence of D17S846, HUM122F4-3′ (accession number L32940) and D17S746. The arabic numbers represent the templates used to perform the PCR amplification of the indicated exon: lane 1, water control; lane 2, 122F4; lane 3, 50H1 P1-phage clones; lane 4, genomic DNA control. The regions of the map in which the entire nucleotide sequence was determined are indicated with left-hand hatch marks.
Figure Legend Snippet: ( A ) A partial map of human chromosome 17q12–q21. The PCR products of an analysis of the P1-phage clones are shown for the presence of exons corresponding to the indicated genes. The arabic numbers represent the templates used to perform the PCR amplification of the indicated exon: lane 1, water control; lane 2, 122F4; lane 3, 50H1 P1-phage clones; lane 4, genomic DNA control. The intron/exon primers of all the genes are available upon request. The sizes of the PCR-amplified fragments are indicated in bp. The arrow indicates transcriptional orientation 5′–3′ of the indicated genes. The portions of the map with right-hand hatch marks correspond to regions in which the nucleotide sequence of the exon/intron junctions was determined. The regions of the map in which the entire nucleotide sequence was determined are indicated with left-hand hatch marks. The centromeric end of the map is on the left and the telomeric end is on the right side. ( B ) A map of the region of chromosome 17q12–q21 affected by LOH. The positions of Exons 1A, 1B, the remainder of JUP exons and HAP1 as well as D17S846, 122F4-3′ and D17S746 on the chromosome map are indicated. The PCR products of an analysis of the P1-phage clones, 50H1 and 122F4, are shown for the presence of D17S846, HUM122F4-3′ (accession number L32940) and D17S746. The arabic numbers represent the templates used to perform the PCR amplification of the indicated exon: lane 1, water control; lane 2, 122F4; lane 3, 50H1 P1-phage clones; lane 4, genomic DNA control. The regions of the map in which the entire nucleotide sequence was determined are indicated with left-hand hatch marks.

Techniques Used: Polymerase Chain Reaction, Clone Assay, Amplification, Sequencing

An RT–PCR assay of JUP Exon 1A ( A ), Exon 1B ( B ) and GAPDH ( C ) RNA expression. Lane 1 is Marker Hae III-digested phi X174 DNA. RT–PCR was performed on RNA from: lane 2, heart; lane 3, kidney; lane 4, peripheral blood lymphocytes; lane 5, liver; lane 6, placenta; lane 7, lung; lane 8, muscle; lane 9, ovary; lane 10, mammary gland; lane 11, small intestine; lane 12, EST-395811 cDNA containing Exon 1B; lane 13, pHPG Ca 2.1 cDNA containing Exon 1A; lane 14, water.
Figure Legend Snippet: An RT–PCR assay of JUP Exon 1A ( A ), Exon 1B ( B ) and GAPDH ( C ) RNA expression. Lane 1 is Marker Hae III-digested phi X174 DNA. RT–PCR was performed on RNA from: lane 2, heart; lane 3, kidney; lane 4, peripheral blood lymphocytes; lane 5, liver; lane 6, placenta; lane 7, lung; lane 8, muscle; lane 9, ovary; lane 10, mammary gland; lane 11, small intestine; lane 12, EST-395811 cDNA containing Exon 1B; lane 13, pHPG Ca 2.1 cDNA containing Exon 1A; lane 14, water.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, RNA Expression, Marker

Related Articles

SYBR Green Assay:

Article Title: IB4-binding sensory neurons in the adult rat express a novel 3′ UTR-extended isoform of CaMK4 that is associated with its localization to axons
Article Snippet: .. PCR reactions were performed with 5ng input cDNA in triplicate using 2X SYBR green master mix (Roche). qPCR was performed with SYBR green detection using a Corbett Rotorgene instrument with the following cycle parameters:1) hotstart for 10 mins at 95C, 2) cycle: 95C for 30 seconds, anneal/extend for 60s at 60C. .. SYBR green fluorescence was determined at the end of each extension.

Article Title: Adjusting microbiome profiles for differences in microbial load by spike-in bacteria
Article Snippet: .. PCR reactions included 1 μM each of eubacterial 16S rRNA gene primers 764 F and 907R (quantification primers) and the LightCycler 480 SYBR Green I Master Kit (Roche). .. Quantification standards were generated by cloning complex PCR amplicon mixtures that were generated from a caecal microbiome DNA preparation of wild type C57BL/6J mice (using primers 341 F and 1061R) into the pGEM-T.Easy vector (Promega, Madison, WI, USA).

Amplification:

Article Title: A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis
Article Snippet: .. Minisatellite PCR amplification and genotyping PCR reactions were performed in 15 μl containing 1 ng of DNA, 1x Long Range Reaction Buffer 3 (Roche-Boehringer), 1 unit of Taq DNA polymerase, 200 μM of each dNTP, 0.3 μM of each flanking primer. .. The Taq DNA polymerase was either prepared essentially as described in [ ] or purchased from Qbiogen or Roche-Boehringer.

Size-exclusion Chromatography:

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: .. The nested PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C for denaturation, 40 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C for final extension. .. RT-PCR Total RNA was isolated from PBMCs or lymph node cells using TRIzol Reagent (Invitrogen, Carlsbad, CA) and RT-PCR was performed using RNA LA PCR Kit (AMV) Ver.

Quantitative RT-PCR:

Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering
Article Snippet: .. The PCR reactions were performed using a qRT-PCR LightCycler® 96 Instrument (Roche, Basel, Swiss), where the total volume per reaction was 10 μl, containing 10 μM of each reference primer (Table ), the corresponding diluted cDNA and 2x FastStart Essential DNA Green Master (Roche). .. Standardisation runs were performed using a LightCycler® 96 thermocycler (Roche, Basel, Swiss), with thermocycling parameters possessing a pre-incubation of 3 min at 95 °C, followed by a three step amplification programme of 40 cycles consisting of a denaturation, annealing and extension step set at 95 °C for 10 s, 60 °C for 15 s and 72 °C for 30 s, respectively.

Article Title: Lanthanide-dependent cross-feeding of methane-derived carbon is linked by microbial community interactions
Article Snippet: .. PCR reactions for qRT-PCR were performed as described in ref. using a LightCycler 2.0 (Roche Diagnostics) and LightCycler Software Version 3.5 (Roche). .. We used the 16S gene to normalize expression values.

Real-time Polymerase Chain Reaction:

Article Title: IB4-binding sensory neurons in the adult rat express a novel 3′ UTR-extended isoform of CaMK4 that is associated with its localization to axons
Article Snippet: .. PCR reactions were performed with 5ng input cDNA in triplicate using 2X SYBR green master mix (Roche). qPCR was performed with SYBR green detection using a Corbett Rotorgene instrument with the following cycle parameters:1) hotstart for 10 mins at 95C, 2) cycle: 95C for 30 seconds, anneal/extend for 60s at 60C. .. SYBR green fluorescence was determined at the end of each extension.

Polymerase Chain Reaction:

Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering
Article Snippet: .. The PCR reactions were performed using a qRT-PCR LightCycler® 96 Instrument (Roche, Basel, Swiss), where the total volume per reaction was 10 μl, containing 10 μM of each reference primer (Table ), the corresponding diluted cDNA and 2x FastStart Essential DNA Green Master (Roche). .. Standardisation runs were performed using a LightCycler® 96 thermocycler (Roche, Basel, Swiss), with thermocycling parameters possessing a pre-incubation of 3 min at 95 °C, followed by a three step amplification programme of 40 cycles consisting of a denaturation, annealing and extension step set at 95 °C for 10 s, 60 °C for 15 s and 72 °C for 30 s, respectively.

Article Title: IB4-binding sensory neurons in the adult rat express a novel 3′ UTR-extended isoform of CaMK4 that is associated with its localization to axons
Article Snippet: .. PCR reactions were performed with 5ng input cDNA in triplicate using 2X SYBR green master mix (Roche). qPCR was performed with SYBR green detection using a Corbett Rotorgene instrument with the following cycle parameters:1) hotstart for 10 mins at 95C, 2) cycle: 95C for 30 seconds, anneal/extend for 60s at 60C. .. SYBR green fluorescence was determined at the end of each extension.

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: .. The PCR reactions were performed using FastStart Taq DNA Polymerase (Roche, Mannheim, Germany). .. The PCR primer pairs and annealing temperatures are shown in Table .

Article Title: A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis
Article Snippet: .. Minisatellite PCR amplification and genotyping PCR reactions were performed in 15 μl containing 1 ng of DNA, 1x Long Range Reaction Buffer 3 (Roche-Boehringer), 1 unit of Taq DNA polymerase, 200 μM of each dNTP, 0.3 μM of each flanking primer. .. The Taq DNA polymerase was either prepared essentially as described in [ ] or purchased from Qbiogen or Roche-Boehringer.

Article Title: Adjusting microbiome profiles for differences in microbial load by spike-in bacteria
Article Snippet: .. PCR reactions included 1 μM each of eubacterial 16S rRNA gene primers 764 F and 907R (quantification primers) and the LightCycler 480 SYBR Green I Master Kit (Roche). .. Quantification standards were generated by cloning complex PCR amplicon mixtures that were generated from a caecal microbiome DNA preparation of wild type C57BL/6J mice (using primers 341 F and 1061R) into the pGEM-T.Easy vector (Promega, Madison, WI, USA).

Article Title: Synergism between DNA methylation and macroH2A1 occupancy in epigenetic silencing of the tumor suppressor gene p16(CDKN2A)
Article Snippet: .. PCR reactions were performed using Faststart taq DNA polymerase (Roche). ..

Article Title: Lanthanide-dependent cross-feeding of methane-derived carbon is linked by microbial community interactions
Article Snippet: .. PCR reactions for qRT-PCR were performed as described in ref. using a LightCycler 2.0 (Roche Diagnostics) and LightCycler Software Version 3.5 (Roche). .. We used the 16S gene to normalize expression values.

Nested PCR:

Article Title: Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms
Article Snippet: .. The nested PCR reactions were performed using FastStart Taq DNA Polymerase (Roche) with the following condition: 5 minutes at 95°C for denaturation, 40 cycles of 30 sec at 95°C, 30 sec at each annealing temperature (Table ), 30 sec at 72°C, and 2 min at 72°C for final extension. .. RT-PCR Total RNA was isolated from PBMCs or lymph node cells using TRIzol Reagent (Invitrogen, Carlsbad, CA) and RT-PCR was performed using RNA LA PCR Kit (AMV) Ver.

Software:

Article Title: Lanthanide-dependent cross-feeding of methane-derived carbon is linked by microbial community interactions
Article Snippet: .. PCR reactions for qRT-PCR were performed as described in ref. using a LightCycler 2.0 (Roche Diagnostics) and LightCycler Software Version 3.5 (Roche). .. We used the 16S gene to normalize expression values.

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  • 90
    Roche real time quantitative reverse transcription pcr rt qpcr reactions
    Quantitative <t>RT-PCR</t> analysis of candidate genes. Mock infected plants were used to normalize fold change of early and late samples in leaf and tuber tissues as indicated. White bars represent the <t>RT-qPCR</t> analysis; grey bars show the microarray data. Error bars represent standard error (SE). L23 and PP2A genes were used as internal normalization controls. Asterisk indicates that the means of fold change of the mock and infected samples are significantly different (Student’s t-test).
    Real Time Quantitative Reverse Transcription Pcr Rt Qpcr Reactions, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche quantitative real time polymerase chain reaction
    Exogenous BDNF increases BDNF/TrkB/SYN mRNA levels of Alzheimer's disease rats. BDNF (A), TrkB (B), and SYN (C) mRNA expression in the rat hippocampus of each group was examined using <t>quantitative</t> <t>polymerase</t> <t>chain</t> <t>reaction.</t> Data are expressed as the mean ± SD (one-way analysis of variance followed by the Bonferroni confidence interval adjustment test). *** P
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    Roche qmsp quantitative methylation specific polymerase chain reaction qmsp
    Comparison of methylation status of PAX1 gene by thiol-labeled AuNPs methods and <t>qMSP.</t> Notes: ( A ) Receiver operating characteristic (ROC) curve analysis by qMSP and ( B and C ) thiol-labeled AuNPs methods. Methylation distribution at various disease stages by histopathology detected by qMSP method. The y-axis is Δ Cp , which represents the <t>DNA</t> methylation level of the PAX1 gene. The dashed line represents the cut-off with a Δ Cp of 10.57. ( D ) Methylated rate distribution at various disease stages detected by thiol-labeled AuNP method. The y-axis is percentage of methylation rate. The dashed line represents the cut-off with a methylated percentage of 31.27% (* P -value
    Qmsp Quantitative Methylation Specific Polymerase Chain Reaction Qmsp, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche quantitative polymerase chain reaction qpcr
    Graphical representation of quantitative <t>PCR</t> results. Total RNA was extracted from control and heat-shocked crustaceans, and Nrhe_Chalamont_Hsp70 mRNA expression was analysed with specific probes. Control animals ( C ; fold induction = 1)
    Quantitative Polymerase Chain Reaction Qpcr, supplied by Roche, used in various techniques. Bioz Stars score: 95/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Quantitative RT-PCR analysis of candidate genes. Mock infected plants were used to normalize fold change of early and late samples in leaf and tuber tissues as indicated. White bars represent the RT-qPCR analysis; grey bars show the microarray data. Error bars represent standard error (SE). L23 and PP2A genes were used as internal normalization controls. Asterisk indicates that the means of fold change of the mock and infected samples are significantly different (Student’s t-test).

    Journal: PLoS ONE

    Article Title: Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection

    doi: 10.1371/journal.pone.0150711

    Figure Lengend Snippet: Quantitative RT-PCR analysis of candidate genes. Mock infected plants were used to normalize fold change of early and late samples in leaf and tuber tissues as indicated. White bars represent the RT-qPCR analysis; grey bars show the microarray data. Error bars represent standard error (SE). L23 and PP2A genes were used as internal normalization controls. Asterisk indicates that the means of fold change of the mock and infected samples are significantly different (Student’s t-test).

    Article Snippet: Three technical replicates of each three biological replicates (for infected tuber tissue only two biological replicates) were used in subsequent Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) reactions using FastStart Universal Probe Master with Rox (Roche Diagnostics, Switzerland).

    Techniques: Quantitative RT-PCR, Infection, Microarray

    Exogenous BDNF increases BDNF/TrkB/SYN mRNA levels of Alzheimer's disease rats. BDNF (A), TrkB (B), and SYN (C) mRNA expression in the rat hippocampus of each group was examined using quantitative polymerase chain reaction. Data are expressed as the mean ± SD (one-way analysis of variance followed by the Bonferroni confidence interval adjustment test). *** P

    Journal: Neural Regeneration Research

    Article Title: Exogenous brain-derived neurotrophic factor attenuates cognitive impairment induced by okadaic acid in a rat model of Alzheimer's disease

    doi: 10.4103/1673-5374.241471

    Figure Lengend Snippet: Exogenous BDNF increases BDNF/TrkB/SYN mRNA levels of Alzheimer's disease rats. BDNF (A), TrkB (B), and SYN (C) mRNA expression in the rat hippocampus of each group was examined using quantitative polymerase chain reaction. Data are expressed as the mean ± SD (one-way analysis of variance followed by the Bonferroni confidence interval adjustment test). *** P

    Article Snippet: Quantitative real-time polymerase chain reaction The fresh hippocampal tissues were put in commercial RNA extraction reagent (Roche Applied Science, Indianapolis, IN, USA) for quantitative polymerase chain reaction (qPCR) analysis.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Comparison of methylation status of PAX1 gene by thiol-labeled AuNPs methods and qMSP. Notes: ( A ) Receiver operating characteristic (ROC) curve analysis by qMSP and ( B and C ) thiol-labeled AuNPs methods. Methylation distribution at various disease stages by histopathology detected by qMSP method. The y-axis is Δ Cp , which represents the DNA methylation level of the PAX1 gene. The dashed line represents the cut-off with a Δ Cp of 10.57. ( D ) Methylated rate distribution at various disease stages detected by thiol-labeled AuNP method. The y-axis is percentage of methylation rate. The dashed line represents the cut-off with a methylated percentage of 31.27% (* P -value

    Journal: International Journal of Nanomedicine

    Article Title: Real-time colorimetric detection of DNA methylation of the PAX1 gene in cervical scrapings for cervical cancer screening with thiol-labeled PCR primers and gold nanoparticles

    doi: 10.2147/IJN.S116288

    Figure Lengend Snippet: Comparison of methylation status of PAX1 gene by thiol-labeled AuNPs methods and qMSP. Notes: ( A ) Receiver operating characteristic (ROC) curve analysis by qMSP and ( B and C ) thiol-labeled AuNPs methods. Methylation distribution at various disease stages by histopathology detected by qMSP method. The y-axis is Δ Cp , which represents the DNA methylation level of the PAX1 gene. The dashed line represents the cut-off with a Δ Cp of 10.57. ( D ) Methylated rate distribution at various disease stages detected by thiol-labeled AuNP method. The y-axis is percentage of methylation rate. The dashed line represents the cut-off with a methylated percentage of 31.27% (* P -value

    Article Snippet: Quantification of DNA methylation through qMSP Quantitative methylation-specific polymerase chain reaction (qMSP) by TaqMan-based technologies was performed in Lightcycler LC480 real-time polymerase chain reaction (PCR) system (Hoffman-La Roche Ltd., Basel, Switzerland) to detect DNA methylation.

    Techniques: Methylation, Labeling, Histopathology, DNA Methylation Assay

    Graphical representation of quantitative PCR results. Total RNA was extracted from control and heat-shocked crustaceans, and Nrhe_Chalamont_Hsp70 mRNA expression was analysed with specific probes. Control animals ( C ; fold induction = 1)

    Journal:

    Article Title: First cellular approach of the effects of global warming on groundwater organisms: a study of the HSP70 gene expression

    doi: 10.1007/s12192-009-0139-4

    Figure Lengend Snippet: Graphical representation of quantitative PCR results. Total RNA was extracted from control and heat-shocked crustaceans, and Nrhe_Chalamont_Hsp70 mRNA expression was analysed with specific probes. Control animals ( C ; fold induction = 1)

    Article Snippet: Quantitative real-time PCR analysis HSP70 gene expression data were quantified using quantitative polymerase chain reaction (qPCR) performed on a LightCycler® sequence analysis system (Roche Diagnostics, Basel, Switzerland) using the QuantiTect SYBR® Green PCR kit (Qiagen). qPCR amplification was performed using synthetic DNA-specific primers corresponding to the transcript of SmRNA that allowed us to determine the reverse transcription efficiency for each sample. qPCR amplification of the gene of interest, HSP70, was then performed using specific primers designed from the alignment of the eight sequences of HSP70 from Niphargus obtained in this study, forward primer F307-PCRQ 5′-GCTGCGATTGCTTACGG-3′ and reverse primer R408-PCRQ 5′-CGCCAGCAGTAGATTTCACCTC-3′.

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Graphical representation of quantitative PCR results. Total RNA was extracted from control and heat-shocked crustaceans, and Nrhe_Chalamont_Hsp70 mRNA expression was analysed with specific probes. Control animals ( C ; fold induction = 1)

    Journal:

    Article Title: First cellular approach of the effects of global warming on groundwater organisms: a study of the HSP70 gene expression

    doi: 10.1007/s12192-009-0139-4

    Figure Lengend Snippet: Graphical representation of quantitative PCR results. Total RNA was extracted from control and heat-shocked crustaceans, and Nrhe_Chalamont_Hsp70 mRNA expression was analysed with specific probes. Control animals ( C ; fold induction = 1)

    Article Snippet: Quantitative real-time PCR analysis HSP70 gene expression data were quantified using quantitative polymerase chain reaction (qPCR) performed on a LightCycler® sequence analysis system (Roche Diagnostics, Basel, Switzerland) using the QuantiTect SYBR® Green PCR kit (Qiagen). qPCR amplification was performed using synthetic DNA-specific primers corresponding to the transcript of SmRNA that allowed us to determine the reverse transcription efficiency for each sample. qPCR amplification of the gene of interest, HSP70, was then performed using specific primers designed from the alignment of the eight sequences of HSP70 from Niphargus obtained in this study, forward primer F307-PCRQ 5′-GCTGCGATTGCTTACGG-3′ and reverse primer R408-PCRQ 5′-CGCCAGCAGTAGATTTCACCTC-3′.

    Techniques: Real-time Polymerase Chain Reaction, Expressing