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Roche bisulfite treated dna
<t>DNA</t> methylation and expression level of the PHD3 gene in HCT116 and DLD-1 CRC cells. A. HCT116 and DLD-1 cells were cultured under normoxic or hypoxic (1% O 2 ) conditions for 48 hrs. Cells were then used for DNA isolation followed by bisulfite modification. Methylation percentage of three DNA fragments within the PHD3 CpG island (Additional file 1 , Additional file 2 ) in HCT116 and DLD-1 cells under hypoxic and normoxic conditions was determined by Real Time <t>PCR</t> amplification of bisulfite treated standard and cell line DNA, followed by comparison of their HRM profiles. B. Cells were cultured in DMEM either in hypoxic (1%O 2 ) or normoxic conditions for 48 hrs. After incubation, the cells were used for total RNA isolation and reverse transcription. The PHD3 cDNA levels were determined by RQ-PCR relative quantification analysis. RQ-PCR results were standardized by the geometric mean of PBGD and hMRPL19 cDNA levels. PHD3 cDNA levels are expressed as a multiplicity of these cDNA copies in the cell line’s calibrator. C. Cells were cultured in DMEM either in hypoxic (1%O 2 ) ( H ) or normoxic ( N ) conditions for 48 hrs. Cells were then used for protein isolation. Proteins were separated by 10% SDS-PAGE, and transferred to a membrane that was then immunoblotted with Rp anti - PHD3 Ab and incubated with goat anti-rabbit HRP-conjugated Ab. The membrane was then stripped and reblotted with Rp anti-GAPDH Ab, followed by incubation with goat anti-rabbit HRP-conjugated Ab. The band densitometry readings were normalized to GAPDH loading control. The ratio of PHD3 to GAPDH for DLD-1 in normoxic conditions was assumed to be 1.
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1) Product Images from "Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer"

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

Journal: BMC Cancer

doi: 10.1186/1471-2407-13-526

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

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

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

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

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

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

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

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

2) Product Images from "A panel of genes methylated with high frequency in colorectal cancer"

Article Title: A panel of genes methylated with high frequency in colorectal cancer

Journal: BMC Cancer

doi: 10.1186/1471-2407-14-54

Profiles of gene methylation for six amplicons. Individual panels show plots of CpG site methylation across the indicated amplicons. Data is presented for 10 individual cancer tissues (red), 10 matched non-neoplastic colon tissues (blue), a 50:50 mix of wbc DNA and fully methylated DNA (green) and wbc blood DNA (ochre). CpG sites are equispaced along the x-axis with labels showing the relative position of each CpG site within the amplicon, relative to the start of the forward primer. Chromosomal locations of amplicons are provided in Additional file 2 : Table S3. The y-axis shows the proportion of methylated cytosines at a CpG site. Sudden coordinated changes in measured methylation rate, such as that at coordinate 134 of the GRASP Region 3 amplicon, is due to a DNA alignment technical artefact caused by long thymine homopolymer repeats creating errors within the pyrosequencing reads.
Figure Legend Snippet: Profiles of gene methylation for six amplicons. Individual panels show plots of CpG site methylation across the indicated amplicons. Data is presented for 10 individual cancer tissues (red), 10 matched non-neoplastic colon tissues (blue), a 50:50 mix of wbc DNA and fully methylated DNA (green) and wbc blood DNA (ochre). CpG sites are equispaced along the x-axis with labels showing the relative position of each CpG site within the amplicon, relative to the start of the forward primer. Chromosomal locations of amplicons are provided in Additional file 2 : Table S3. The y-axis shows the proportion of methylated cytosines at a CpG site. Sudden coordinated changes in measured methylation rate, such as that at coordinate 134 of the GRASP Region 3 amplicon, is due to a DNA alignment technical artefact caused by long thymine homopolymer repeats creating errors within the pyrosequencing reads.

Techniques Used: Methylation, Amplification

Frequency of gene methylation in colorectal neoplasia. Methylation levels of individual genes (left hand labels) were determined by qMSP using primer pairs and conditions described in Additional file 2 : Table S4. The percentage of samples showing greater than 10% methylation is shown for CRC (red spots), matched normal tissue (green) and adenomas (purple). Up to 78 cancer samples were tested for any individual gene. The size of the spots is proportional to a log2 transformation of the number of samples tested (small gray circle10; medium gray circle 40; large gray circle 80). The difference in detection cycle between CpGenome™ DNA and wbc DNA (ΔCt ) is presented as bars to the right with lengths proportional to the ΔCt value (which is also presented numerically within each bar). An asterix denotes the qMSP reaction completed before reaction products from wbc DNA were detected, so the ΔCt is at least this value.
Figure Legend Snippet: Frequency of gene methylation in colorectal neoplasia. Methylation levels of individual genes (left hand labels) were determined by qMSP using primer pairs and conditions described in Additional file 2 : Table S4. The percentage of samples showing greater than 10% methylation is shown for CRC (red spots), matched normal tissue (green) and adenomas (purple). Up to 78 cancer samples were tested for any individual gene. The size of the spots is proportional to a log2 transformation of the number of samples tested (small gray circle10; medium gray circle 40; large gray circle 80). The difference in detection cycle between CpGenome™ DNA and wbc DNA (ΔCt ) is presented as bars to the right with lengths proportional to the ΔCt value (which is also presented numerically within each bar). An asterix denotes the qMSP reaction completed before reaction products from wbc DNA were detected, so the ΔCt is at least this value.

Techniques Used: Methylation, Transformation Assay

3) Product Images from "DNA methylation and histone modifications regulate SOX11 expression in lymphoid and solid cancer cells"

Article Title: DNA methylation and histone modifications regulate SOX11 expression in lymphoid and solid cancer cells

Journal: BMC Cancer

doi: 10.1186/s12885-015-1208-y

Correlation between SOX11 promoter methylation and expression. The correlation between DNA methylation and gene expression was analyzed with RT-qPCR and western blot. In RT-qPCR, CT values > 35 were considered below detection limit and corresponding SOX11 levels were set to zero. (A) MethyLight (x-axis) and melt curve analysis (see filled, grey or open diamonds) showed an inverse correlation between SOX11 mRNA and promoter methylation in lymphoma cell lines. (B) Likewise, inverse correlation between SOX11 mRNA and promoter methylation was seen for solid cancer cell lines. For clarity, SOX11 positive (western blot) cell line names are shaded. (C) Western blot analysis of SOX11 and GAPDH in Burkitt’s lymphoma, follicular lymphoma, diffuse large B-cell lymphoma and mantle cell lymphoma cell-lines. (D) Western blot analysis of SOX11 and GAPDH in breast cancer, ovarian cancer, lung cancer, brain cancer and neuroblastoma cell lines.
Figure Legend Snippet: Correlation between SOX11 promoter methylation and expression. The correlation between DNA methylation and gene expression was analyzed with RT-qPCR and western blot. In RT-qPCR, CT values > 35 were considered below detection limit and corresponding SOX11 levels were set to zero. (A) MethyLight (x-axis) and melt curve analysis (see filled, grey or open diamonds) showed an inverse correlation between SOX11 mRNA and promoter methylation in lymphoma cell lines. (B) Likewise, inverse correlation between SOX11 mRNA and promoter methylation was seen for solid cancer cell lines. For clarity, SOX11 positive (western blot) cell line names are shaded. (C) Western blot analysis of SOX11 and GAPDH in Burkitt’s lymphoma, follicular lymphoma, diffuse large B-cell lymphoma and mantle cell lymphoma cell-lines. (D) Western blot analysis of SOX11 and GAPDH in breast cancer, ovarian cancer, lung cancer, brain cancer and neuroblastoma cell lines.

Techniques Used: Methylation, Expressing, DNA Methylation Assay, Quantitative RT-PCR, Western Blot

4) Product Images from "A panel of genes methylated with high frequency in colorectal cancer"

Article Title: A panel of genes methylated with high frequency in colorectal cancer

Journal: BMC Cancer

doi: 10.1186/1471-2407-14-54

Profiles of gene methylation for six amplicons. Individual panels show plots of CpG site methylation across the indicated amplicons. Data is presented for 10 individual cancer tissues (red), 10 matched non-neoplastic colon tissues (blue), a 50:50 mix of wbc DNA and fully methylated DNA (green) and wbc blood DNA (ochre). CpG sites are equispaced along the x-axis with labels showing the relative position of each CpG site within the amplicon, relative to the start of the forward primer. Chromosomal locations of amplicons are provided in Additional file 2 : Table S3. The y-axis shows the proportion of methylated cytosines at a CpG site. Sudden coordinated changes in measured methylation rate, such as that at coordinate 134 of the GRASP Region 3 amplicon, is due to a DNA alignment technical artefact caused by long thymine homopolymer repeats creating errors within the pyrosequencing reads.
Figure Legend Snippet: Profiles of gene methylation for six amplicons. Individual panels show plots of CpG site methylation across the indicated amplicons. Data is presented for 10 individual cancer tissues (red), 10 matched non-neoplastic colon tissues (blue), a 50:50 mix of wbc DNA and fully methylated DNA (green) and wbc blood DNA (ochre). CpG sites are equispaced along the x-axis with labels showing the relative position of each CpG site within the amplicon, relative to the start of the forward primer. Chromosomal locations of amplicons are provided in Additional file 2 : Table S3. The y-axis shows the proportion of methylated cytosines at a CpG site. Sudden coordinated changes in measured methylation rate, such as that at coordinate 134 of the GRASP Region 3 amplicon, is due to a DNA alignment technical artefact caused by long thymine homopolymer repeats creating errors within the pyrosequencing reads.

Techniques Used: Methylation, Amplification

Frequency of gene methylation in colorectal neoplasia. Methylation levels of individual genes (left hand labels) were determined by qMSP using primer pairs and conditions described in Additional file 2 : Table S4. The percentage of samples showing greater than 10% methylation is shown for CRC (red spots), matched normal tissue (green) and adenomas (purple). Up to 78 cancer samples were tested for any individual gene. The size of the spots is proportional to a log2 transformation of the number of samples tested (small gray circle10; medium gray circle 40; large gray circle 80). The difference in detection cycle between CpGenome™ DNA and wbc DNA (ΔCt ) is presented as bars to the right with lengths proportional to the ΔCt value (which is also presented numerically within each bar). An asterix denotes the qMSP reaction completed before reaction products from wbc DNA were detected, so the ΔCt is at least this value.
Figure Legend Snippet: Frequency of gene methylation in colorectal neoplasia. Methylation levels of individual genes (left hand labels) were determined by qMSP using primer pairs and conditions described in Additional file 2 : Table S4. The percentage of samples showing greater than 10% methylation is shown for CRC (red spots), matched normal tissue (green) and adenomas (purple). Up to 78 cancer samples were tested for any individual gene. The size of the spots is proportional to a log2 transformation of the number of samples tested (small gray circle10; medium gray circle 40; large gray circle 80). The difference in detection cycle between CpGenome™ DNA and wbc DNA (ΔCt ) is presented as bars to the right with lengths proportional to the ΔCt value (which is also presented numerically within each bar). An asterix denotes the qMSP reaction completed before reaction products from wbc DNA were detected, so the ΔCt is at least this value.

Techniques Used: Methylation, Transformation Assay

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

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

Journal: Journal of Cancer Research and Clinical Oncology

doi: 10.1007/s00432-014-1901-2

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

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

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

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

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

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

The Kaplan–Meier survival analysis among patients with colorectal cancer according to the TET1 mRNA level ( a ), TET1 DNA methylation ( b ), TET2 ( c ) and TET3 ( d ) mRNA level. Patients were subdivided into three groups: low, intermediate, and high for each TET1, TET2 and TET3 transcript levels in histopathologically unchanged and cancerous tissue or DNA methylation absent/present in TET1 promoter in cancerous tissue. p values for overall survival (OS) and disease-free survival (DFS) were determined with the log rank test and given only for significant results. n , number of patients
Figure Legend Snippet: The Kaplan–Meier survival analysis among patients with colorectal cancer according to the TET1 mRNA level ( a ), TET1 DNA methylation ( b ), TET2 ( c ) and TET3 ( d ) mRNA level. Patients were subdivided into three groups: low, intermediate, and high for each TET1, TET2 and TET3 transcript levels in histopathologically unchanged and cancerous tissue or DNA methylation absent/present in TET1 promoter in cancerous tissue. p values for overall survival (OS) and disease-free survival (DFS) were determined with the log rank test and given only for significant results. n , number of patients

Techniques Used: DNA Methylation Assay

6) Product Images from "DNA Methylation Influences miRNA Expression in Gonadotroph Pituitary Tumors"

Article Title: DNA Methylation Influences miRNA Expression in Gonadotroph Pituitary Tumors

Journal: Life

doi: 10.3390/life10050059

DNA methylation and relative expression of selected miRNA genes in the validation cohort. ( A ) Difference in DNA methylation at miRNA-encoding gene promoters in gonadotroph PitNET samples and normal pituitaries. Each dot represents the average methylation level of CpGs covered by PCR amplicon in a particular sample; mean values are horizontal lines. ( B ) Matched result of measurements of DNA methylation at particular CpG sites and relative miRNA expression levels. Each dot represents the CpG methylation/miRNA expression of individual PitNET sample.
Figure Legend Snippet: DNA methylation and relative expression of selected miRNA genes in the validation cohort. ( A ) Difference in DNA methylation at miRNA-encoding gene promoters in gonadotroph PitNET samples and normal pituitaries. Each dot represents the average methylation level of CpGs covered by PCR amplicon in a particular sample; mean values are horizontal lines. ( B ) Matched result of measurements of DNA methylation at particular CpG sites and relative miRNA expression levels. Each dot represents the CpG methylation/miRNA expression of individual PitNET sample.

Techniques Used: DNA Methylation Assay, Expressing, Methylation, Polymerase Chain Reaction, Amplification, CpG Methylation Assay

Related Articles

Amplification:

Article Title: A panel of genes methylated with high frequency in colorectal cancer
Article Snippet: .. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. .. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP).

Article Title: Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer
Article Snippet: .. DNA methylation assessment by high resolution melting (HRM) analysis Methylation levels of DNA fragments located within the CpG island of the PHD1 , PHD2 , PHD3 and FIH genes (Additional file ) were determined by Real Time PCR amplification of bisulfite treated DNA followed by HRM profile analysis by Light Cycler®480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany). .. For PCR amplification, 1 μl of the bisulfite treated DNA from patients, HCT116, DLD-1 cells, or standards, and primers (Additional file , Additional file ) was added to 19 μl of 5 X Hot FIREPol EvaGreen HRM Mix, Solis BioDyne Co. (Tartu, Estonia).

Article Title: DNA Methylation Influences miRNA Expression in Gonadotroph Pituitary Tumors
Article Snippet: .. Bisulfite-treated DNA was PCR (polymerase chain reaction) -amplified in 30 µL volume containing 2 mM MgCl2, 0.25 mM dNTPs, 0.2 μM of each primer, and 0.5 U of FastStart DNA Polymerase (Roche Applied Science, Mannheim, Germany) with following temperature cycles: 94 °C for 3 min; 40 cycles of 30 s at 94 °C. .. Then, PCR amplicons were purified and analyzed using the PyroMark Q24 System (Qiagen) according to manufacturer’s protocol.

Article Title: Clinical significance of DNA methylation mRNA levels of TET family members in colorectal cancer
Article Snippet: .. DNA methylation assessment by high-resolution melting (HRM) analysis The methylation levels of DNA fragments located within the CpG islands of the TET1 , TET2 and TET3 genes were determined by real-time PCR amplification of bisulfite-treated DNA, followed by HRM profile analysis by Light Cycler® 480 or LightCycler® 96 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany). .. For PCR amplification, 1 μl of either the bisulfite-treated DNA from patients or standards, together with primers (Supplementary Table 1), was added to 19 μl of 5 X Hot FIREPol EvaGreen HRM Mix, Solis BioDyne Co. (Tartu, Estonia).

Methylation:

Article Title: Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer
Article Snippet: .. DNA methylation assessment by high resolution melting (HRM) analysis Methylation levels of DNA fragments located within the CpG island of the PHD1 , PHD2 , PHD3 and FIH genes (Additional file ) were determined by Real Time PCR amplification of bisulfite treated DNA followed by HRM profile analysis by Light Cycler®480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany). .. For PCR amplification, 1 μl of the bisulfite treated DNA from patients, HCT116, DLD-1 cells, or standards, and primers (Additional file , Additional file ) was added to 19 μl of 5 X Hot FIREPol EvaGreen HRM Mix, Solis BioDyne Co. (Tartu, Estonia).

Article Title: A panel of genes methylated with high frequency in colorectal cancer
Article Snippet: .. A total of 59 conversion specific PCRs across 27 genes in triplicate (primers and PCR conditions described in Additional file and Additional file : Table S3) were applied to 5-10 ng bisulfite treated DNA including, peripheral blood lymphocyte DNA (wbc DNA, Roche Applied Science, Cat # 1 691 112) and a 1:1 mix of wbc DNA and enzymatically methylated DNA (CpGenome™ Methylated DNA, Millipore). ..

Article Title: Clinical significance of DNA methylation mRNA levels of TET family members in colorectal cancer
Article Snippet: .. DNA methylation assessment by high-resolution melting (HRM) analysis The methylation levels of DNA fragments located within the CpG islands of the TET1 , TET2 and TET3 genes were determined by real-time PCR amplification of bisulfite-treated DNA, followed by HRM profile analysis by Light Cycler® 480 or LightCycler® 96 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany). .. For PCR amplification, 1 μl of either the bisulfite-treated DNA from patients or standards, together with primers (Supplementary Table 1), was added to 19 μl of 5 X Hot FIREPol EvaGreen HRM Mix, Solis BioDyne Co. (Tartu, Estonia).

DNA Methylation Assay:

Article Title: Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer
Article Snippet: .. DNA methylation assessment by high resolution melting (HRM) analysis Methylation levels of DNA fragments located within the CpG island of the PHD1 , PHD2 , PHD3 and FIH genes (Additional file ) were determined by Real Time PCR amplification of bisulfite treated DNA followed by HRM profile analysis by Light Cycler®480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany). .. For PCR amplification, 1 μl of the bisulfite treated DNA from patients, HCT116, DLD-1 cells, or standards, and primers (Additional file , Additional file ) was added to 19 μl of 5 X Hot FIREPol EvaGreen HRM Mix, Solis BioDyne Co. (Tartu, Estonia).

Article Title: Clinical significance of DNA methylation mRNA levels of TET family members in colorectal cancer
Article Snippet: .. DNA methylation assessment by high-resolution melting (HRM) analysis The methylation levels of DNA fragments located within the CpG islands of the TET1 , TET2 and TET3 genes were determined by real-time PCR amplification of bisulfite-treated DNA, followed by HRM profile analysis by Light Cycler® 480 or LightCycler® 96 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany). .. For PCR amplification, 1 μl of either the bisulfite-treated DNA from patients or standards, together with primers (Supplementary Table 1), was added to 19 μl of 5 X Hot FIREPol EvaGreen HRM Mix, Solis BioDyne Co. (Tartu, Estonia).

Real-time Polymerase Chain Reaction:

Article Title: Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer
Article Snippet: .. DNA methylation assessment by high resolution melting (HRM) analysis Methylation levels of DNA fragments located within the CpG island of the PHD1 , PHD2 , PHD3 and FIH genes (Additional file ) were determined by Real Time PCR amplification of bisulfite treated DNA followed by HRM profile analysis by Light Cycler®480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany). .. For PCR amplification, 1 μl of the bisulfite treated DNA from patients, HCT116, DLD-1 cells, or standards, and primers (Additional file , Additional file ) was added to 19 μl of 5 X Hot FIREPol EvaGreen HRM Mix, Solis BioDyne Co. (Tartu, Estonia).

Article Title: Clinical significance of DNA methylation mRNA levels of TET family members in colorectal cancer
Article Snippet: .. DNA methylation assessment by high-resolution melting (HRM) analysis The methylation levels of DNA fragments located within the CpG islands of the TET1 , TET2 and TET3 genes were determined by real-time PCR amplification of bisulfite-treated DNA, followed by HRM profile analysis by Light Cycler® 480 or LightCycler® 96 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany). .. For PCR amplification, 1 μl of either the bisulfite-treated DNA from patients or standards, together with primers (Supplementary Table 1), was added to 19 μl of 5 X Hot FIREPol EvaGreen HRM Mix, Solis BioDyne Co. (Tartu, Estonia).

Polymerase Chain Reaction:

Article Title: A panel of genes methylated with high frequency in colorectal cancer
Article Snippet: .. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. .. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP).

Article Title: Methylation Status of Transcriptional Modulatory Genes Associated with Colorectal Cancer in Northeast China
Article Snippet: .. Each reaction mixture contained 0.5 mL of bisulfite-treated DNA, 0.1 mL of each forward and reverse primer (10 mM), 2.5 mL of LightCycler 480 High Resolution Melting Master (Roche), 0.6 mL MgCl2 (25 mM) and 1.2 mL polymerase chain reaction (PCR)-grade water to a total volume of 5 mL. .. Universal unmethylated (0% methylated) and methylated (100% methylated) human whole genomic DNA samples (Zymo Research Corp., Irvine, CA, USA) were used as the control and calibrator samples, respectively.

Article Title: DNA Methylation Influences miRNA Expression in Gonadotroph Pituitary Tumors
Article Snippet: .. Bisulfite-treated DNA was PCR (polymerase chain reaction) -amplified in 30 µL volume containing 2 mM MgCl2, 0.25 mM dNTPs, 0.2 μM of each primer, and 0.5 U of FastStart DNA Polymerase (Roche Applied Science, Mannheim, Germany) with following temperature cycles: 94 °C for 3 min; 40 cycles of 30 s at 94 °C. .. Then, PCR amplicons were purified and analyzed using the PyroMark Q24 System (Qiagen) according to manufacturer’s protocol.

Article Title: A panel of genes methylated with high frequency in colorectal cancer
Article Snippet: .. A total of 59 conversion specific PCRs across 27 genes in triplicate (primers and PCR conditions described in Additional file and Additional file : Table S3) were applied to 5-10 ng bisulfite treated DNA including, peripheral blood lymphocyte DNA (wbc DNA, Roche Applied Science, Cat # 1 691 112) and a 1:1 mix of wbc DNA and enzymatically methylated DNA (CpGenome™ Methylated DNA, Millipore). ..

Sequencing:

Article Title: A panel of genes methylated with high frequency in colorectal cancer
Article Snippet: .. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. .. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP).

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    Roche bisulfite treated dna
    <t>DNA</t> methylation and expression level of the PHD3 gene in HCT116 and DLD-1 CRC cells. A. HCT116 and DLD-1 cells were cultured under normoxic or hypoxic (1% O 2 ) conditions for 48 hrs. Cells were then used for DNA isolation followed by bisulfite modification. Methylation percentage of three DNA fragments within the PHD3 CpG island (Additional file 1 , Additional file 2 ) in HCT116 and DLD-1 cells under hypoxic and normoxic conditions was determined by Real Time <t>PCR</t> amplification of bisulfite treated standard and cell line DNA, followed by comparison of their HRM profiles. B. Cells were cultured in DMEM either in hypoxic (1%O 2 ) or normoxic conditions for 48 hrs. After incubation, the cells were used for total RNA isolation and reverse transcription. The PHD3 cDNA levels were determined by RQ-PCR relative quantification analysis. RQ-PCR results were standardized by the geometric mean of PBGD and hMRPL19 cDNA levels. PHD3 cDNA levels are expressed as a multiplicity of these cDNA copies in the cell line’s calibrator. C. Cells were cultured in DMEM either in hypoxic (1%O 2 ) ( H ) or normoxic ( N ) conditions for 48 hrs. Cells were then used for protein isolation. Proteins were separated by 10% SDS-PAGE, and transferred to a membrane that was then immunoblotted with Rp anti - PHD3 Ab and incubated with goat anti-rabbit HRP-conjugated Ab. The membrane was then stripped and reblotted with Rp anti-GAPDH Ab, followed by incubation with goat anti-rabbit HRP-conjugated Ab. The band densitometry readings were normalized to GAPDH loading control. The ratio of PHD3 to GAPDH for DLD-1 in normoxic conditions was assumed to be 1.
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    DNA methylation and expression level of the PHD3 gene in HCT116 and DLD-1 CRC cells. A. HCT116 and DLD-1 cells were cultured under normoxic or hypoxic (1% O 2 ) conditions for 48 hrs. Cells were then used for DNA isolation followed by bisulfite modification. Methylation percentage of three DNA fragments within the PHD3 CpG island (Additional file 1 , Additional file 2 ) in HCT116 and DLD-1 cells under hypoxic and normoxic conditions was determined by Real Time PCR amplification of bisulfite treated standard and cell line DNA, followed by comparison of their HRM profiles. B. Cells were cultured in DMEM either in hypoxic (1%O 2 ) or normoxic conditions for 48 hrs. After incubation, the cells were used for total RNA isolation and reverse transcription. The PHD3 cDNA levels were determined by RQ-PCR relative quantification analysis. RQ-PCR results were standardized by the geometric mean of PBGD and hMRPL19 cDNA levels. PHD3 cDNA levels are expressed as a multiplicity of these cDNA copies in the cell line’s calibrator. C. Cells were cultured in DMEM either in hypoxic (1%O 2 ) ( H ) or normoxic ( N ) conditions for 48 hrs. Cells were then used for protein isolation. Proteins were separated by 10% SDS-PAGE, and transferred to a membrane that was then immunoblotted with Rp anti - PHD3 Ab and incubated with goat anti-rabbit HRP-conjugated Ab. The membrane was then stripped and reblotted with Rp anti-GAPDH Ab, followed by incubation with goat anti-rabbit HRP-conjugated Ab. The band densitometry readings were normalized to GAPDH loading control. The ratio of PHD3 to GAPDH for DLD-1 in normoxic conditions was assumed to be 1.

    Journal: BMC Cancer

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

    doi: 10.1186/1471-2407-13-526

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

    Article Snippet: DNA methylation assessment by high resolution melting (HRM) analysis Methylation levels of DNA fragments located within the CpG island of the PHD1 , PHD2 , PHD3 and FIH genes (Additional file ) were determined by Real Time PCR amplification of bisulfite treated DNA followed by HRM profile analysis by Light Cycler®480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany).

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

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

    Journal: BMC Cancer

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

    doi: 10.1186/1471-2407-13-526

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

    Article Snippet: DNA methylation assessment by high resolution melting (HRM) analysis Methylation levels of DNA fragments located within the CpG island of the PHD1 , PHD2 , PHD3 and FIH genes (Additional file ) were determined by Real Time PCR amplification of bisulfite treated DNA followed by HRM profile analysis by Light Cycler®480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany).

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

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

    Journal: BMC Cancer

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

    doi: 10.1186/1471-2407-13-526

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

    Article Snippet: DNA methylation assessment by high resolution melting (HRM) analysis Methylation levels of DNA fragments located within the CpG island of the PHD1 , PHD2 , PHD3 and FIH genes (Additional file ) were determined by Real Time PCR amplification of bisulfite treated DNA followed by HRM profile analysis by Light Cycler®480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany).

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

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

    Journal: BMC Cancer

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

    doi: 10.1186/1471-2407-13-526

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

    Article Snippet: DNA methylation assessment by high resolution melting (HRM) analysis Methylation levels of DNA fragments located within the CpG island of the PHD1 , PHD2 , PHD3 and FIH genes (Additional file ) were determined by Real Time PCR amplification of bisulfite treated DNA followed by HRM profile analysis by Light Cycler®480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany).

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

    Profiles of gene methylation for six amplicons. Individual panels show plots of CpG site methylation across the indicated amplicons. Data is presented for 10 individual cancer tissues (red), 10 matched non-neoplastic colon tissues (blue), a 50:50 mix of wbc DNA and fully methylated DNA (green) and wbc blood DNA (ochre). CpG sites are equispaced along the x-axis with labels showing the relative position of each CpG site within the amplicon, relative to the start of the forward primer. Chromosomal locations of amplicons are provided in Additional file 2 : Table S3. The y-axis shows the proportion of methylated cytosines at a CpG site. Sudden coordinated changes in measured methylation rate, such as that at coordinate 134 of the GRASP Region 3 amplicon, is due to a DNA alignment technical artefact caused by long thymine homopolymer repeats creating errors within the pyrosequencing reads.

    Journal: BMC Cancer

    Article Title: A panel of genes methylated with high frequency in colorectal cancer

    doi: 10.1186/1471-2407-14-54

    Figure Lengend Snippet: Profiles of gene methylation for six amplicons. Individual panels show plots of CpG site methylation across the indicated amplicons. Data is presented for 10 individual cancer tissues (red), 10 matched non-neoplastic colon tissues (blue), a 50:50 mix of wbc DNA and fully methylated DNA (green) and wbc blood DNA (ochre). CpG sites are equispaced along the x-axis with labels showing the relative position of each CpG site within the amplicon, relative to the start of the forward primer. Chromosomal locations of amplicons are provided in Additional file 2 : Table S3. The y-axis shows the proportion of methylated cytosines at a CpG site. Sudden coordinated changes in measured methylation rate, such as that at coordinate 134 of the GRASP Region 3 amplicon, is due to a DNA alignment technical artefact caused by long thymine homopolymer repeats creating errors within the pyrosequencing reads.

    Article Snippet: A total of 59 conversion specific PCRs across 27 genes in triplicate (primers and PCR conditions described in Additional file and Additional file : Table S3) were applied to 5-10 ng bisulfite treated DNA including, peripheral blood lymphocyte DNA (wbc DNA, Roche Applied Science, Cat # 1 691 112) and a 1:1 mix of wbc DNA and enzymatically methylated DNA (CpGenome™ Methylated DNA, Millipore).

    Techniques: Methylation, Amplification

    Frequency of gene methylation in colorectal neoplasia. Methylation levels of individual genes (left hand labels) were determined by qMSP using primer pairs and conditions described in Additional file 2 : Table S4. The percentage of samples showing greater than 10% methylation is shown for CRC (red spots), matched normal tissue (green) and adenomas (purple). Up to 78 cancer samples were tested for any individual gene. The size of the spots is proportional to a log2 transformation of the number of samples tested (small gray circle10; medium gray circle 40; large gray circle 80). The difference in detection cycle between CpGenome™ DNA and wbc DNA (ΔCt ) is presented as bars to the right with lengths proportional to the ΔCt value (which is also presented numerically within each bar). An asterix denotes the qMSP reaction completed before reaction products from wbc DNA were detected, so the ΔCt is at least this value.

    Journal: BMC Cancer

    Article Title: A panel of genes methylated with high frequency in colorectal cancer

    doi: 10.1186/1471-2407-14-54

    Figure Lengend Snippet: Frequency of gene methylation in colorectal neoplasia. Methylation levels of individual genes (left hand labels) were determined by qMSP using primer pairs and conditions described in Additional file 2 : Table S4. The percentage of samples showing greater than 10% methylation is shown for CRC (red spots), matched normal tissue (green) and adenomas (purple). Up to 78 cancer samples were tested for any individual gene. The size of the spots is proportional to a log2 transformation of the number of samples tested (small gray circle10; medium gray circle 40; large gray circle 80). The difference in detection cycle between CpGenome™ DNA and wbc DNA (ΔCt ) is presented as bars to the right with lengths proportional to the ΔCt value (which is also presented numerically within each bar). An asterix denotes the qMSP reaction completed before reaction products from wbc DNA were detected, so the ΔCt is at least this value.

    Article Snippet: A total of 59 conversion specific PCRs across 27 genes in triplicate (primers and PCR conditions described in Additional file and Additional file : Table S3) were applied to 5-10 ng bisulfite treated DNA including, peripheral blood lymphocyte DNA (wbc DNA, Roche Applied Science, Cat # 1 691 112) and a 1:1 mix of wbc DNA and enzymatically methylated DNA (CpGenome™ Methylated DNA, Millipore).

    Techniques: Methylation, Transformation Assay