bisulfite treated dna  (Qiagen)

 
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    Name:
    EpiTect Bisulfite Kit
    Description:
    For complete bisulfite conversion and cleanup of DNA for methylation analysis Kit contents Qiagen EpiTect Bisulfite Kit 48 Spin Columns 1 to 2g Sample Blood FFPE Tissue DNA Samples Spin Column and 96 well Plate Format Manual Processing 99 4 to 99 8 Conversion Efficiency Ideal for Multiplex PCR Real time PCR Sequencing For Complete Bisulfite Conversion and Cleanup of DNA for Methylation Analysis Includes 48 EpiTect Bisulfite Spin Columns Reaction Mix DNA Protect Buffer Carrier RNA Buffers Benefits Streamlined procedure for fast and reliable results Complete cytosine conversion Unique DNA protection system for highly sensitive analysis Optimized protocols including conversion from FFPE DNA Spin column and 96 well plate formats for high through
    Catalog Number:
    59104
    Price:
    253
    Category:
    EpiTect Bisulfite Kits
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    Structured Review

    Qiagen bisulfite treated dna
    EpiTect Bisulfite Kit
    For complete bisulfite conversion and cleanup of DNA for methylation analysis Kit contents Qiagen EpiTect Bisulfite Kit 48 Spin Columns 1 to 2g Sample Blood FFPE Tissue DNA Samples Spin Column and 96 well Plate Format Manual Processing 99 4 to 99 8 Conversion Efficiency Ideal for Multiplex PCR Real time PCR Sequencing For Complete Bisulfite Conversion and Cleanup of DNA for Methylation Analysis Includes 48 EpiTect Bisulfite Spin Columns Reaction Mix DNA Protect Buffer Carrier RNA Buffers Benefits Streamlined procedure for fast and reliable results Complete cytosine conversion Unique DNA protection system for highly sensitive analysis Optimized protocols including conversion from FFPE DNA Spin column and 96 well plate formats for high through
    https://www.bioz.com/result/bisulfite treated dna/product/Qiagen
    Average 99 stars, based on 167 article reviews
    Price from $9.99 to $1999.99
    bisulfite treated dna - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "MGMT promoter methylation in gliomas-assessment by pyrosequencing and quantitative methylation-specific PCR"

    Article Title: MGMT promoter methylation in gliomas-assessment by pyrosequencing and quantitative methylation-specific PCR

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-10-36

    Gene expression level of MGMT is associated with promoter DNA methylation status . MGMT gene expression in methylated (blue box plots) and unmethylated (red box plots) tissue samples analyzed by two different primer/probe sets (Assay 1; Hs00172470_m1 and assay 2; Hs01037698_m1). ( A ) Methylation status based on results from qMSP. ( B ) Methylation status based on results from pyrosequencing. Abbreviation: qMSP, quantitative methylation specific PCR
    Figure Legend Snippet: Gene expression level of MGMT is associated with promoter DNA methylation status . MGMT gene expression in methylated (blue box plots) and unmethylated (red box plots) tissue samples analyzed by two different primer/probe sets (Assay 1; Hs00172470_m1 and assay 2; Hs01037698_m1). ( A ) Methylation status based on results from qMSP. ( B ) Methylation status based on results from pyrosequencing. Abbreviation: qMSP, quantitative methylation specific PCR

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

    2) Product Images from "Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death"

    Article Title: Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094591

    Primer selection and analytical performance of methylation-specific PCR. A) Schematic illustration of the human INS gene promoter region showing the position of the nine CpG sites. Solid arrows represent the bisulfite-specific primers (BSPs) that amplify both methylated and unmethylated DNA. Dashed arrows represent methylation-specific primers (MSPs) that amplify unmethylated DNA only. B) Unmethylated plasmid was serially diluted after bisulfite conversion and analyzed by qMSP using selected primer sets. Agarose gel electrophoresis of MSP reactions showing the size of the PCR products. C) Graphs of real-time SYBR Green PCR data showing linearity of C q versus log copy number of unmethylated plasmid (averages and standard deviation (SD)) from 5 to 10 6 copies.
    Figure Legend Snippet: Primer selection and analytical performance of methylation-specific PCR. A) Schematic illustration of the human INS gene promoter region showing the position of the nine CpG sites. Solid arrows represent the bisulfite-specific primers (BSPs) that amplify both methylated and unmethylated DNA. Dashed arrows represent methylation-specific primers (MSPs) that amplify unmethylated DNA only. B) Unmethylated plasmid was serially diluted after bisulfite conversion and analyzed by qMSP using selected primer sets. Agarose gel electrophoresis of MSP reactions showing the size of the PCR products. C) Graphs of real-time SYBR Green PCR data showing linearity of C q versus log copy number of unmethylated plasmid (averages and standard deviation (SD)) from 5 to 10 6 copies.

    Techniques Used: Selection, Methylation, Polymerase Chain Reaction, Plasmid Preparation, Agarose Gel Electrophoresis, SYBR Green Assay, Standard Deviation

    Beta cell specificity of MSP. Serial dilutions of the bisulfite-converted gDNA obtained from human islets, blood, spleen and colon were used as a template for nested PCR using either BSP (A) or MSP (B) in the first-step reaction. The products were used as a template for the second-step MSP reaction. The data display the mean ± SEM of the Relative Unmethylation Ratio (RUR). The cloned INS promoter was used for normalization and standardization of the results. Statistically significant differences at each DNA concentration between islets and other tissues were calculated using two way ANOVA and the significance level indicated by asterisks (****, p
    Figure Legend Snippet: Beta cell specificity of MSP. Serial dilutions of the bisulfite-converted gDNA obtained from human islets, blood, spleen and colon were used as a template for nested PCR using either BSP (A) or MSP (B) in the first-step reaction. The products were used as a template for the second-step MSP reaction. The data display the mean ± SEM of the Relative Unmethylation Ratio (RUR). The cloned INS promoter was used for normalization and standardization of the results. Statistically significant differences at each DNA concentration between islets and other tissues were calculated using two way ANOVA and the significance level indicated by asterisks (****, p

    Techniques Used: Nested PCR, Clone Assay, Concentration Assay

    3) Product Images from "Bi-PROF"

    Article Title: Bi-PROF

    Journal: Epigenetics

    doi: 10.4161/epi.25242

    Figure 1. Schematic experimental workflow Bi-PROF. As an example we show the generation of a set of amplicons for 3 different sample DNAs. Amplicons are generated with DNA sample-specific tags (so called MIDs). The sample and amplicon complexity
    Figure Legend Snippet: Figure 1. Schematic experimental workflow Bi-PROF. As an example we show the generation of a set of amplicons for 3 different sample DNAs. Amplicons are generated with DNA sample-specific tags (so called MIDs). The sample and amplicon complexity

    Techniques Used: Generated, Amplification

    4) Product Images from "Prolonged re-expression of the hypermethylated gene EPB41L3 using artificial transcription factors and epigenetic drugs"

    Article Title: Prolonged re-expression of the hypermethylated gene EPB41L3 using artificial transcription factors and epigenetic drugs

    Journal: Epigenetics

    doi: 10.1080/15592294.2015.1034415

    Targeted reversal of histone marks by EPB41L3- ATFs, but moderate binding specificity. Association of 21ab-NoEf with its targeted site as analyzed by an HA tag ChIP ( A ) and ChIP-Seq ( B ) for the 21ab-ZFP and an empty vector in EPB41L3 methylated CaSki cells. For the ChIP-Seq, the local coverage is shown for a region of 20 kb as visualized by R using the coverage distribution report obtained from NextGENe. Indicated is the ZFP target site (▴). ( C ) Representation of the number of identified ChIP-Seq peaks vs. the average coverage per peak [shown for peaks with a coverage of four or more (maximum 100)] determined by the Peak Identification Report obtained from NextGENe. ( D ) Percentage of ChIP-Seq peaks (with a coverage of 10 or more) bound to promoters, genes or other regions (such as noncoding DNA). ( E ) Graphical representation of peaks with a coverage of 10 or more bound to promoter regions. ( F ) Change in histone marks after re-expression of EPB41L3 in methylated CaSki (left) and unmethylated C33A cells (right). A quantitative ChIP for H3Ac and H3K9me3 was performed after treatment with 21ab-VP64 and/or 21ab-NoEf. Values represent the mean percentage of input of three independent experiments ± SEM, (HA tag one experiment).
    Figure Legend Snippet: Targeted reversal of histone marks by EPB41L3- ATFs, but moderate binding specificity. Association of 21ab-NoEf with its targeted site as analyzed by an HA tag ChIP ( A ) and ChIP-Seq ( B ) for the 21ab-ZFP and an empty vector in EPB41L3 methylated CaSki cells. For the ChIP-Seq, the local coverage is shown for a region of 20 kb as visualized by R using the coverage distribution report obtained from NextGENe. Indicated is the ZFP target site (▴). ( C ) Representation of the number of identified ChIP-Seq peaks vs. the average coverage per peak [shown for peaks with a coverage of four or more (maximum 100)] determined by the Peak Identification Report obtained from NextGENe. ( D ) Percentage of ChIP-Seq peaks (with a coverage of 10 or more) bound to promoters, genes or other regions (such as noncoding DNA). ( E ) Graphical representation of peaks with a coverage of 10 or more bound to promoter regions. ( F ) Change in histone marks after re-expression of EPB41L3 in methylated CaSki (left) and unmethylated C33A cells (right). A quantitative ChIP for H3Ac and H3K9me3 was performed after treatment with 21ab-VP64 and/or 21ab-NoEf. Values represent the mean percentage of input of three independent experiments ± SEM, (HA tag one experiment).

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Plasmid Preparation, Methylation, Expressing

    5) Product Images from "Development of a multiplex methylation-specific PCR as candidate triage test for women with an HPV-positive cervical scrape"

    Article Title: Development of a multiplex methylation-specific PCR as candidate triage test for women with an HPV-positive cervical scrape

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-12-551

    Standard curves of CADM1 and MAL in a multiplex qMSP with ACTB * tested on a serial dilution series of methylation positive SiHa DNA in methylation negative DNA of primary keratinocytes. Standard curve of CADM1 ( A ) and MAL ( B ) with the previously described primers 9 . CADM1 ( C ) and MAL ( D ) primers with nearly identical annealing temperatures as ACTB primers. For the dilution series the PCR efficiencies were calculated by E=(10 (−1/Slope) -1)×100%. The qPCR efficiencies increased from 76% to 87% for CADM1 and 52% to 105% for MAL. * ) Multiplex qMSP was performed with an ACTB primer concentration of 200 nM.
    Figure Legend Snippet: Standard curves of CADM1 and MAL in a multiplex qMSP with ACTB * tested on a serial dilution series of methylation positive SiHa DNA in methylation negative DNA of primary keratinocytes. Standard curve of CADM1 ( A ) and MAL ( B ) with the previously described primers 9 . CADM1 ( C ) and MAL ( D ) primers with nearly identical annealing temperatures as ACTB primers. For the dilution series the PCR efficiencies were calculated by E=(10 (−1/Slope) -1)×100%. The qPCR efficiencies increased from 76% to 87% for CADM1 and 52% to 105% for MAL. * ) Multiplex qMSP was performed with an ACTB primer concentration of 200 nM.

    Techniques Used: Multiplex Assay, Serial Dilution, Methylation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Concentration Assay

    High linearity between singleplex and multiplex analysis on serial dilutions of methylated DNA (SiHa) in unmethylated DNA (EK). The multiplex reactions showed comparable efficiencies as the singleplex reactions for CADM1 ( A ), MAL ( B ) and hsa-miR-124-2 ( C ). Same thresholds were used in singleplex and multiplex qMSP, resulting in different Cq values for all targets. When adjusting the thresholds, the same Cq values can be obtained, resulting in overlap of the standard curves.
    Figure Legend Snippet: High linearity between singleplex and multiplex analysis on serial dilutions of methylated DNA (SiHa) in unmethylated DNA (EK). The multiplex reactions showed comparable efficiencies as the singleplex reactions for CADM1 ( A ), MAL ( B ) and hsa-miR-124-2 ( C ). Same thresholds were used in singleplex and multiplex qMSP, resulting in different Cq values for all targets. When adjusting the thresholds, the same Cq values can be obtained, resulting in overlap of the standard curves.

    Techniques Used: Multiplex Assay, Methylation

    6) Product Images from "HERV-K and LINE-1 DNA Methylation and Reexpression in Urothelial Carcinoma"

    Article Title: HERV-K and LINE-1 DNA Methylation and Reexpression in Urothelial Carcinoma

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2013.00255

    DNA methylation and expression changes of LINE-1 elements in bladder cancer . (A) DNA methylation in the CpG islets of LINE-1 was quantified by pyrosequencing in a set of 10 normal urothelial cell cultures and 18 bladder cancer cell lines. For comparison, LINE-1 DNA methylation was assessed in immortalized urothelial cells (TERT-NHUC) and in uncultured epithelial cells (uncultured UP) and connective tissue from one ureter. (B) LINE-1 RNA levels from the 5′- and 3′-regions were measured by qRT-PCR in a set of 6 normal urothelial cell cultures and 18 bladder cancer cell lines. Inset: amplification of different retroelements ( HERVK17 , LINE-1_5 ′ and LINE-1_3 ′) was measured in three bladder cancer cell lines using cDNA preparations with (+) or without (−) reverse transcriptase (RT) to assess the impact of genomic DNA contamination. Results were adjusted for each assay and cell line to reverse transcriptase positive preparations set as 100% (C) LINE-1 DNA methylation and expression of the 5′- and 3′-regions were analyzed in a set of 12 benign and 23 cancerous bladder tissues or 11 benign and 24 tumorous bladder tissues, respectively. Methylation is plotted as mean methylation value from four CpGs in percent (A,C) . RNA levels were each normalized to TBP and standardized to either the median RNA level of normal urothelial cell cultures (B) or the median RNA level of benign bladder tissues (C) set as 1. p Values calculated by the Mann–Whitney U -test were given above the brackets for significant changes ( p
    Figure Legend Snippet: DNA methylation and expression changes of LINE-1 elements in bladder cancer . (A) DNA methylation in the CpG islets of LINE-1 was quantified by pyrosequencing in a set of 10 normal urothelial cell cultures and 18 bladder cancer cell lines. For comparison, LINE-1 DNA methylation was assessed in immortalized urothelial cells (TERT-NHUC) and in uncultured epithelial cells (uncultured UP) and connective tissue from one ureter. (B) LINE-1 RNA levels from the 5′- and 3′-regions were measured by qRT-PCR in a set of 6 normal urothelial cell cultures and 18 bladder cancer cell lines. Inset: amplification of different retroelements ( HERVK17 , LINE-1_5 ′ and LINE-1_3 ′) was measured in three bladder cancer cell lines using cDNA preparations with (+) or without (−) reverse transcriptase (RT) to assess the impact of genomic DNA contamination. Results were adjusted for each assay and cell line to reverse transcriptase positive preparations set as 100% (C) LINE-1 DNA methylation and expression of the 5′- and 3′-regions were analyzed in a set of 12 benign and 23 cancerous bladder tissues or 11 benign and 24 tumorous bladder tissues, respectively. Methylation is plotted as mean methylation value from four CpGs in percent (A,C) . RNA levels were each normalized to TBP and standardized to either the median RNA level of normal urothelial cell cultures (B) or the median RNA level of benign bladder tissues (C) set as 1. p Values calculated by the Mann–Whitney U -test were given above the brackets for significant changes ( p

    Techniques Used: DNA Methylation Assay, Expressing, Quantitative RT-PCR, Amplification, Methylation, MANN-WHITNEY

    7) Product Images from "Nicotiana Small RNA Sequences Support a Host Genome Origin of Cucumber Mosaic Virus Satellite RNA"

    Article Title: Nicotiana Small RNA Sequences Support a Host Genome Origin of Cucumber Mosaic Virus Satellite RNA

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004906

    Methylation analysis of the 35S-GUS:sSat transgene in transgenic N. tabacum using McrBC PCR. (A) and (B) McrBC PCR of 35S promoter, different regions of GUS coding sequence, and Y-Sat. (C) qPCR quantification of McrBC digestion of the Y-Sat sequence shown in (B) (nt. 1-214) (left) and MUG assay of GUS activity in the transgenic plants used for the McrBC PCR analysis (right). M, DNA size marker.
    Figure Legend Snippet: Methylation analysis of the 35S-GUS:sSat transgene in transgenic N. tabacum using McrBC PCR. (A) and (B) McrBC PCR of 35S promoter, different regions of GUS coding sequence, and Y-Sat. (C) qPCR quantification of McrBC digestion of the Y-Sat sequence shown in (B) (nt. 1-214) (left) and MUG assay of GUS activity in the transgenic plants used for the McrBC PCR analysis (right). M, DNA size marker.

    Techniques Used: Methylation, Transgenic Assay, Polymerase Chain Reaction, Sequencing, Real-time Polymerase Chain Reaction, Mug Assay, Activity Assay, Marker

    8) Product Images from "Histopathological and clonal study of combined lobular and ductal carcinoma of the breast"

    Article Title: Histopathological and clonal study of combined lobular and ductal carcinoma of the breast

    Journal: Pathology International

    doi: 10.1111/pin.12065

    Clonal analysis with X-chromosome inactivation patterns by methylation-specific PCR on the HUMARA (human androgen receptor) gene. The bisulfite-treated DNA was amplified with PCR in use of methylated and unmethylated primer pairs. (a) A random X chromosome inactivation (heterozygous) pattern is shown in control female blood cells. (b) In contrast, a non-random inactivation (homozygous) pattern is shown in male blood cells. (c) The PCR products of case no. 2 are displayed as a random inactivation (heterozygous) pattern in the non-tumor tissue, and as a non-random inactivation (homozygous) pattern in both (d) the lobular carcinoma component and (e) the ductal carcinoma component. (A left-sided minor peak detected in ductal component is quite less dominant than the main peak and accessed as an artifact.)
    Figure Legend Snippet: Clonal analysis with X-chromosome inactivation patterns by methylation-specific PCR on the HUMARA (human androgen receptor) gene. The bisulfite-treated DNA was amplified with PCR in use of methylated and unmethylated primer pairs. (a) A random X chromosome inactivation (heterozygous) pattern is shown in control female blood cells. (b) In contrast, a non-random inactivation (homozygous) pattern is shown in male blood cells. (c) The PCR products of case no. 2 are displayed as a random inactivation (heterozygous) pattern in the non-tumor tissue, and as a non-random inactivation (homozygous) pattern in both (d) the lobular carcinoma component and (e) the ductal carcinoma component. (A left-sided minor peak detected in ductal component is quite less dominant than the main peak and accessed as an artifact.)

    Techniques Used: Methylation, Polymerase Chain Reaction, Amplification

    9) Product Images from "Quantitative Evaluation of DNA Methylation Patterns for ALVE and TVB Genes in a Neoplastic Disease Susceptible and Resistant Chicken Model"

    Article Title: Quantitative Evaluation of DNA Methylation Patterns for ALVE and TVB Genes in a Neoplastic Disease Susceptible and Resistant Chicken Model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001731

    Results of quantitative DNA methylation analysis of inbred lines 6 3 and 7 2 in promoter region of TVB in spleen, liver, hypothalamus and blood. n = 5 for each line. ** P
    Figure Legend Snippet: Results of quantitative DNA methylation analysis of inbred lines 6 3 and 7 2 in promoter region of TVB in spleen, liver, hypothalamus and blood. n = 5 for each line. ** P

    Techniques Used: DNA Methylation Assay

    The results of DNA methylation analysis in a unique region of ALVE 1 from blood in line 7 2 , line 6 3 and RCS C, F, J, L, M and T blood. n = 5 for each line or strain. P > 0.05.
    Figure Legend Snippet: The results of DNA methylation analysis in a unique region of ALVE 1 from blood in line 7 2 , line 6 3 and RCS C, F, J, L, M and T blood. n = 5 for each line or strain. P > 0.05.

    Techniques Used: DNA Methylation Assay

    DNA sequencing of PPT region and partial U3 region of 3′ LTR in ALVE between line 6 3 and line 7 2 . Brown box shows the PPT region. Brown arrow shows one variation located on PPT region changed from G in line 6 3 to A in line 7 2 . Black arrow shows another variation located in U3 region of 3′ LTR changed from T in line 6 3 to C in line 7 2 . n = 11 for each line.
    Figure Legend Snippet: DNA sequencing of PPT region and partial U3 region of 3′ LTR in ALVE between line 6 3 and line 7 2 . Brown box shows the PPT region. Brown arrow shows one variation located on PPT region changed from G in line 6 3 to A in line 7 2 . Black arrow shows another variation located in U3 region of 3′ LTR changed from T in line 6 3 to C in line 7 2 . n = 11 for each line.

    Techniques Used: DNA Sequencing

    Results of quantitative DNA methylation analysis in promoter region of TVB in blood. Line 6 3 and 7 2 are two parental lines. C, F, J, L, M and T are six recombinant congenic strains (RCS) chicken. n = 5 for each line and RCS. * P
    Figure Legend Snippet: Results of quantitative DNA methylation analysis in promoter region of TVB in blood. Line 6 3 and 7 2 are two parental lines. C, F, J, L, M and T are six recombinant congenic strains (RCS) chicken. n = 5 for each line and RCS. * P

    Techniques Used: DNA Methylation Assay, Recombinant

    Regression analysis of mRNA expression level of PPT-U3 region of ALVE and DNA methylation contents of ALVE region2.
    Figure Legend Snippet: Regression analysis of mRNA expression level of PPT-U3 region of ALVE and DNA methylation contents of ALVE region2.

    Techniques Used: Expressing, DNA Methylation Assay

    10) Product Images from "Methylome analysis of extreme chemoresponsive patients identifies novel markers of platinum sensitivity in high-grade serous ovarian cancer"

    Article Title: Methylome analysis of extreme chemoresponsive patients identifies novel markers of platinum sensitivity in high-grade serous ovarian cancer

    Journal: BMC Medicine

    doi: 10.1186/s12916-017-0870-0

    FZD10 is an epigenetically regulated gene by DNA methylation. a Correlation analysis of average methylation as determined by bisulfite pyrosequencing and relative mRNA level of FZD10 in external cohort patients ( n = 32) showed significant inverse correlation between methylation and their correspondent expression using Pearson correlation testing. b qRT-PCR of FZD10 was performed to determine relative mRNA levels in responder ( n = 10) and non-responder HGSOC patient groups ( n = 22). Heatmaps show average methylation percentage ( c ) and relative mRNA expression ( d ) of FZD10 in various ovarian cancer cell lines ( n = 11), treated with or without DAC for 72 h (DAC + or –). Most cell lines show DAC-induced demethylation (from blue to dark red, change in methylation percentage) with subsequent upregulation of mRNA (from black to green, relative fold expression). Relative gene expression of FZD10 (∆Ct = Ct FZD10 – Ct GAPDH ) for each untreated cell line is mentioned in front of the heatmap
    Figure Legend Snippet: FZD10 is an epigenetically regulated gene by DNA methylation. a Correlation analysis of average methylation as determined by bisulfite pyrosequencing and relative mRNA level of FZD10 in external cohort patients ( n = 32) showed significant inverse correlation between methylation and their correspondent expression using Pearson correlation testing. b qRT-PCR of FZD10 was performed to determine relative mRNA levels in responder ( n = 10) and non-responder HGSOC patient groups ( n = 22). Heatmaps show average methylation percentage ( c ) and relative mRNA expression ( d ) of FZD10 in various ovarian cancer cell lines ( n = 11), treated with or without DAC for 72 h (DAC + or –). Most cell lines show DAC-induced demethylation (from blue to dark red, change in methylation percentage) with subsequent upregulation of mRNA (from black to green, relative fold expression). Relative gene expression of FZD10 (∆Ct = Ct FZD10 – Ct GAPDH ) for each untreated cell line is mentioned in front of the heatmap

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

    11) Product Images from "Differential MSH2 promoter methylation in blood cells of Neurofibromatosis type 1 (NF1) patients"

    Article Title: Differential MSH2 promoter methylation in blood cells of Neurofibromatosis type 1 (NF1) patients

    Journal:

    doi: 10.1038/ejhg.2009.129

    Methylation-specific PCR (MSP). Left side: A 20 bp ladder was used as the standard for gel analysis. U – unmethylated (primers to detect unmethylated DNA) and M – methylated (primers to detect methylated DNA). Samples 1–4
    Figure Legend Snippet: Methylation-specific PCR (MSP). Left side: A 20 bp ladder was used as the standard for gel analysis. U – unmethylated (primers to detect unmethylated DNA) and M – methylated (primers to detect methylated DNA). Samples 1–4

    Techniques Used: Methylation, Polymerase Chain Reaction

    12) Product Images from "Rapid analysis of CpG methylation patterns using RNase T1 cleavage and MALDI-TOF"

    Article Title: Rapid analysis of CpG methylation patterns using RNase T1 cleavage and MALDI-TOF

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh165

    Direct analysis of two bisulfite-converted colon tumor DNA samples (T1 and T2) and two normal colon DNA samples (N1 and N2), respectively, by PCR, in vitro transcription, RNAse T1 cleavage and MALDI-TOF in comparison with DNA mixtures with defined methylation states. (top, mass spectrum of samples T1, T2, N1, N2, respectively; bottom, spectra of DNA mixtures with different, defined methylation states and signal intensity of control fragment C1 set as maximum, fragment numbers given for 100%).
    Figure Legend Snippet: Direct analysis of two bisulfite-converted colon tumor DNA samples (T1 and T2) and two normal colon DNA samples (N1 and N2), respectively, by PCR, in vitro transcription, RNAse T1 cleavage and MALDI-TOF in comparison with DNA mixtures with defined methylation states. (top, mass spectrum of samples T1, T2, N1, N2, respectively; bottom, spectra of DNA mixtures with different, defined methylation states and signal intensity of control fragment C1 set as maximum, fragment numbers given for 100%).

    Techniques Used: Polymerase Chain Reaction, In Vitro, Methylation

    ( I ) Multiple alignment of the virtual sequences of the investigated T7 transcripts derived from the bisulfite sequence traces of: PCR product from methylated, bisulfite-treated DNA (A), PCR product from unmethylated, bisulfite-treated DNA (B), PCR product from plasmid pEPI2383 DNA (C) and plasmid pEPI2383 without further PCR (D). Guanosines derived from originally methylated cytosines are highlighted in red, from non-converted cytosines in blue and from the residual vector, T7 Promoter and control tag in yellow. Gene-specific priming sites are marked in italic. ( II ) Fragmentation (sequences, their related m/z values and positions of the represented CpG) of the T7 transcript A (in I). Fragments from the T7 domain of the primer are tagged with P1–P4, gene-specific fragments are labeled with numbers (1–14) and fragments from the attached control tag are marked with C1–C2. Fragments size > 11000 Da and
    Figure Legend Snippet: ( I ) Multiple alignment of the virtual sequences of the investigated T7 transcripts derived from the bisulfite sequence traces of: PCR product from methylated, bisulfite-treated DNA (A), PCR product from unmethylated, bisulfite-treated DNA (B), PCR product from plasmid pEPI2383 DNA (C) and plasmid pEPI2383 without further PCR (D). Guanosines derived from originally methylated cytosines are highlighted in red, from non-converted cytosines in blue and from the residual vector, T7 Promoter and control tag in yellow. Gene-specific priming sites are marked in italic. ( II ) Fragmentation (sequences, their related m/z values and positions of the represented CpG) of the T7 transcript A (in I). Fragments from the T7 domain of the primer are tagged with P1–P4, gene-specific fragments are labeled with numbers (1–14) and fragments from the attached control tag are marked with C1–C2. Fragments size > 11000 Da and

    Techniques Used: Derivative Assay, Sequencing, Polymerase Chain Reaction, Methylation, Plasmid Preparation, Labeling

    13) Product Images from "5-Azacytidine Enhances the Radiosensitivity of CNE2 and SUNE1 Cells In Vitro and In Vivo Possibly by Altering DNA Methylation"

    Article Title: 5-Azacytidine Enhances the Radiosensitivity of CNE2 and SUNE1 Cells In Vitro and In Vivo Possibly by Altering DNA Methylation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093273

    Effect of 5-azaC on the DNA methylation and expression of representative tumor suppressor genes that are hypermethylated and silenced in NPC. SUNE1 and CNE2 cells and mice bearing CNE2 tumor xenografts were treated with or without 5-azaC. (A) DNA methylation pyrograms for RASSF1A and RPRM in CNE2 cells. (B) Mean levels of DNA methylation for RASSF1A, RPRM, CDKN2A and 14-3-3σ (Student’s t -test, * p
    Figure Legend Snippet: Effect of 5-azaC on the DNA methylation and expression of representative tumor suppressor genes that are hypermethylated and silenced in NPC. SUNE1 and CNE2 cells and mice bearing CNE2 tumor xenografts were treated with or without 5-azaC. (A) DNA methylation pyrograms for RASSF1A and RPRM in CNE2 cells. (B) Mean levels of DNA methylation for RASSF1A, RPRM, CDKN2A and 14-3-3σ (Student’s t -test, * p

    Techniques Used: DNA Methylation Assay, Expressing, Mouse Assay

    14) Product Images from "Genome-wide DNA methylation profiling reveals novel epigenetic signatures in squamous cell lung cancer"

    Article Title: Genome-wide DNA methylation profiling reveals novel epigenetic signatures in squamous cell lung cancer

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4223-3

    Validation of selected methylation biomarkers. a Clinical validation of DNA methylation levels of selected genes in paired LUSC and adjacent NTL tissue by using pyrosequencing. b Clinical validation of mRNA expression of selected genes in paired LUSC and adjacent NTL tissue by using qRT-PCR. c ROC curves and area under the curve (AUC) for the candidate genes. Sensitivity, Specificity and the optimal cut-off values were marked in the figures. *** corresponds to p
    Figure Legend Snippet: Validation of selected methylation biomarkers. a Clinical validation of DNA methylation levels of selected genes in paired LUSC and adjacent NTL tissue by using pyrosequencing. b Clinical validation of mRNA expression of selected genes in paired LUSC and adjacent NTL tissue by using qRT-PCR. c ROC curves and area under the curve (AUC) for the candidate genes. Sensitivity, Specificity and the optimal cut-off values were marked in the figures. *** corresponds to p

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

    15) Product Images from "CpG Island Tumor Suppressor Promoter Methylation in Non-BRCA-Associated Early Mammary Carcinogenesis"

    Article Title: CpG Island Tumor Suppressor Promoter Methylation in Non-BRCA-Associated Early Mammary Carcinogenesis

    Journal: Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology

    doi: 10.1158/1055-9965.EPI-08-0875

    Comparison of Sequenom methylation analysis with conventional MSP. Seven RPFNA samples as well as methylated and unmethylated control DNA samples were tested for methylation status by Sequenom and conventional MSP. Equal amounts (1 μg) of bisulfite-converted genomic DNA were used for each analysis. The sensitivity of Sequenom is 5%, whereas our conventional MSP assay is 0.01%. MC , methylated control; UC , unmethylated control. For MSP: blue columns , presence of methylation; white columns , absence of methylation.
    Figure Legend Snippet: Comparison of Sequenom methylation analysis with conventional MSP. Seven RPFNA samples as well as methylated and unmethylated control DNA samples were tested for methylation status by Sequenom and conventional MSP. Equal amounts (1 μg) of bisulfite-converted genomic DNA were used for each analysis. The sensitivity of Sequenom is 5%, whereas our conventional MSP assay is 0.01%. MC , methylated control; UC , unmethylated control. For MSP: blue columns , presence of methylation; white columns , absence of methylation.

    Techniques Used: Methylation, MSP Assay

    16) Product Images from "Quantitative Analysis and Diagnostic Significance of Methylated SLC19A3 DNA in the Plasma of Breast and Gastric Cancer Patients"

    Article Title: Quantitative Analysis and Diagnostic Significance of Methylated SLC19A3 DNA in the Plasma of Breast and Gastric Cancer Patients

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022233

    Quantitative analysis of plasma methylated SLC19A3 DNA on a group (n = 165) of plasma samples by MSRED-qPCR. Scatter plots of plasma levels of methylated SLC19A3 DNA in 60 healthy normal subjects, 45 gastric cancer (GC) and 60 breast cancer (BC) patients. Plasma level of methylated SLC19A3 DNA is expressed as 2 ΔCt(undigest-digest) . ΔCt (undigest-digest) is calculated by subtracting the Ct values of digested plasma DNA from the Ct values of undigested plasma DNA. Since Ct of undigest should be ≤Ct of digest, the expression level is ranging from 1 to 0. The horizontal black lines denote the means. The blue errors bars denote the ± standard deviations (SD). Statistically significant differences were determined using Mann-Whitney U tests, P
    Figure Legend Snippet: Quantitative analysis of plasma methylated SLC19A3 DNA on a group (n = 165) of plasma samples by MSRED-qPCR. Scatter plots of plasma levels of methylated SLC19A3 DNA in 60 healthy normal subjects, 45 gastric cancer (GC) and 60 breast cancer (BC) patients. Plasma level of methylated SLC19A3 DNA is expressed as 2 ΔCt(undigest-digest) . ΔCt (undigest-digest) is calculated by subtracting the Ct values of digested plasma DNA from the Ct values of undigested plasma DNA. Since Ct of undigest should be ≤Ct of digest, the expression level is ranging from 1 to 0. The horizontal black lines denote the means. The blue errors bars denote the ± standard deviations (SD). Statistically significant differences were determined using Mann-Whitney U tests, P

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction, Expressing, MANN-WHITNEY

    Increased percentage of SLC19A3 DNA methylation in primary breast cancer tissues. Percentage of SLC19A3 promoter methylation between tumor tissues and their paired adjacent non-tumor breast tissues from the 15 breast cancer patients by MS-qPCR. Percentage of methylation in tissue samples was calculated by the following equation: % meth = 100/[1+2 ΔCt(meth-unmeth) ]%. ΔCt (meth-unmeth) was calculated by subtracting the Ct values of methylated SLC19A3 signal from the Ct values of umnethylated SLC19A3 signal. Statistical difference was analyzed by Wilcoxon test, P
    Figure Legend Snippet: Increased percentage of SLC19A3 DNA methylation in primary breast cancer tissues. Percentage of SLC19A3 promoter methylation between tumor tissues and their paired adjacent non-tumor breast tissues from the 15 breast cancer patients by MS-qPCR. Percentage of methylation in tissue samples was calculated by the following equation: % meth = 100/[1+2 ΔCt(meth-unmeth) ]%. ΔCt (meth-unmeth) was calculated by subtracting the Ct values of methylated SLC19A3 signal from the Ct values of umnethylated SLC19A3 signal. Statistical difference was analyzed by Wilcoxon test, P

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

    17) Product Images from "Epigenetic silencing of long non-coding RNA BM742401 in multiple myeloma: impact on prognosis and myeloma dissemination"

    Article Title: Epigenetic silencing of long non-coding RNA BM742401 in multiple myeloma: impact on prognosis and myeloma dissemination

    Journal: Cancer Cell International

    doi: 10.1186/s12935-020-01504-4

    Methylation of BM742401 in healthy controls and HMCLs. a Direct sequencing of M-MSP products from positive control with methylated DNA showed the conversion of all unmethylated cytosines into uracils (turned into thymidines after PCR) but all methylated cytosines remained unchanged, indicating complete bisulfite conversion and specificity of MSP. b M-MSP and U-MSP showed that all healthy controls (N1-N17) were completely unmethylated (UU), whereas positive control with methylated DNA was completely methylated (MM). c M-MSP and U-MSP showed BM742401 was MM in HMCLs, including KMS-12-PE, MOLP-8 and OCI-MY5, partially methylated (MU) in JJN-3, LP-1, OPM-2, U-266, WL-2, OPM-2/BTZ and RPMI-8226R, UU in NCI-H929, RPMI-8226, MMKKF, MMLAL and KMS-11/BTZ
    Figure Legend Snippet: Methylation of BM742401 in healthy controls and HMCLs. a Direct sequencing of M-MSP products from positive control with methylated DNA showed the conversion of all unmethylated cytosines into uracils (turned into thymidines after PCR) but all methylated cytosines remained unchanged, indicating complete bisulfite conversion and specificity of MSP. b M-MSP and U-MSP showed that all healthy controls (N1-N17) were completely unmethylated (UU), whereas positive control with methylated DNA was completely methylated (MM). c M-MSP and U-MSP showed BM742401 was MM in HMCLs, including KMS-12-PE, MOLP-8 and OCI-MY5, partially methylated (MU) in JJN-3, LP-1, OPM-2, U-266, WL-2, OPM-2/BTZ and RPMI-8226R, UU in NCI-H929, RPMI-8226, MMKKF, MMLAL and KMS-11/BTZ

    Techniques Used: Methylation, Sequencing, Positive Control, Polymerase Chain Reaction

    18) Product Images from "Aging-like Spontaneous Epigenetic Silencing Facilitates Wnt Activation, Stemness, and BrafV600E-Induced Tumorigenesis"

    Article Title: Aging-like Spontaneous Epigenetic Silencing Facilitates Wnt Activation, Stemness, and BrafV600E-Induced Tumorigenesis

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2019.01.005

    DNA Methylation Modifications Accrue during Braf V600E -Driven Transformation (A) Bar plot showing numbers of genes identified as differentially methylated In Braf CA 3 and Braf CA-IND 3 compared with Braf EV 3. Enrichment of Wnt-pathway genes in the genes hypermethylated in Braf CA-IND 3 is indicated. (B) Representative CGI (red bars under gene structure) promoter methylation profiles of candidate genes (orange peaks indicate enrichment of reads indicating relative methylation levels). Curved arrows indicate transcription start sites and direction of gene transcription. In the Cdkn2a locus, p16 Ink4a and p19 Arf promoters are shown separately. (C) Heatmap showing validation of CIMP phenotype in the Braf CA-IND at key candidate genes by quantitative methylation-specific PCR (MSP) and bisulfite pyrosequencing. Organoids shown are those that were cultured for 5 months. .
    Figure Legend Snippet: DNA Methylation Modifications Accrue during Braf V600E -Driven Transformation (A) Bar plot showing numbers of genes identified as differentially methylated In Braf CA 3 and Braf CA-IND 3 compared with Braf EV 3. Enrichment of Wnt-pathway genes in the genes hypermethylated in Braf CA-IND 3 is indicated. (B) Representative CGI (red bars under gene structure) promoter methylation profiles of candidate genes (orange peaks indicate enrichment of reads indicating relative methylation levels). Curved arrows indicate transcription start sites and direction of gene transcription. In the Cdkn2a locus, p16 Ink4a and p19 Arf promoters are shown separately. (C) Heatmap showing validation of CIMP phenotype in the Braf CA-IND at key candidate genes by quantitative methylation-specific PCR (MSP) and bisulfite pyrosequencing. Organoids shown are those that were cultured for 5 months. .

    Techniques Used: DNA Methylation Assay, Transformation Assay, Methylation, Polymerase Chain Reaction, Cell Culture

    Long-Term-Cultured Organoids Accumulate CpG-Island DNA Methylation and Show Differentiation Defects (A) DNA methylation accumulation determined by bisulfite pyrosequencing of selected CGI regions in Braf EV 1 and 3 organoids cultured for 2 or 12–14 months and Braf CA-IND 1–3 organoids cultured for 5 months. The suffix “m” in Braf EV 1–12m and Braf EV 3–14m indicates the duration in months for which the organoids were cultured. Whiskers indicate mean (cross bar) ± SD. (B) Representative images showing the growth of long-term-cultured (12–14 months) wild-type Braf EV organoids in medium deficient in indicated ligands, or in medium with all ligands (Full). Results are representative of two experiments performed in duplicate. (C) Quantitative real-time PCR analysis of markers and key cell fate regulators of colon epithelial cells between long-term-cultured (12or14 months) and young (2 months) wild-type Braf EV 1 and 3 organoids. mRNA expression of long-term- relative to short-term-cultured organoids is shown. Error bars, ±SD (n = 3 wells). (D) Projection of confocal images showing enterocyte cell marker Krt20 (green) and proliferating cells (EdU, red) in Braf EV 1 and 3 organoids cultured for the indicated lengths of time. DAPI (blue) is used as a nuclear stain. .
    Figure Legend Snippet: Long-Term-Cultured Organoids Accumulate CpG-Island DNA Methylation and Show Differentiation Defects (A) DNA methylation accumulation determined by bisulfite pyrosequencing of selected CGI regions in Braf EV 1 and 3 organoids cultured for 2 or 12–14 months and Braf CA-IND 1–3 organoids cultured for 5 months. The suffix “m” in Braf EV 1–12m and Braf EV 3–14m indicates the duration in months for which the organoids were cultured. Whiskers indicate mean (cross bar) ± SD. (B) Representative images showing the growth of long-term-cultured (12–14 months) wild-type Braf EV organoids in medium deficient in indicated ligands, or in medium with all ligands (Full). Results are representative of two experiments performed in duplicate. (C) Quantitative real-time PCR analysis of markers and key cell fate regulators of colon epithelial cells between long-term-cultured (12or14 months) and young (2 months) wild-type Braf EV 1 and 3 organoids. mRNA expression of long-term- relative to short-term-cultured organoids is shown. Error bars, ±SD (n = 3 wells). (D) Projection of confocal images showing enterocyte cell marker Krt20 (green) and proliferating cells (EdU, red) in Braf EV 1 and 3 organoids cultured for the indicated lengths of time. DAPI (blue) is used as a nuclear stain. .

    Techniques Used: Cell Culture, DNA Methylation Assay, Real-time Polymerase Chain Reaction, Expressing, Marker, Staining

    Related Articles

    Modification:

    Article Title: Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing
    Article Snippet: .. The kits compared were the Premium Bisulfite kit (Cat. No. C02030030, Diagenode), the EpiTect Bisulfite kit (Cat. No. 59104, Qiagen), the MethylEdge Bisulfite Conversion System (Cat. No. N1301, Promega) and the BisulFlash DNA Modification kit (Cat. No. 1026, Epigentek). .. The performance was tested by using fully methylated and fully unmethylated λ-DNA controls in a series of spikes, by means of Sanger sequencing, Next-Generation Sequencing (NGS), gel electrophoresis and fluorometry.

    Article Title: Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing
    Article Snippet: .. The kits that were used were: the Premium Bisulfite kit (Cat. No. C02030030, Diagenode), the EpiTect Bisulfite kit (Cat. No. 59104, Qiagen), the MethylEdge Bisulfite Conversion System (Cat. No. N1301, Promega) and the BisulFlash DNA Modification kit (Cat. No. 1026, Epigentek). .. For the determination of the DNA yield after bisulfite conversion, the Qubit fluorometer (Invitrogen) was used according to manufacturer's instructions.

    Article Title: Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells
    Article Snippet: .. Methylation Specific polymerase chain reaction (MSP-PCR) A total of 1 μg of DNA extracted from total (parental) DU145 and LNCaP cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. .. PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic or bisulfite treated DNA.

    Polymerase Chain Reaction:

    Article Title: Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells
    Article Snippet: .. Methylation Specific polymerase chain reaction (MSP-PCR) A total of 1 μg of DNA extracted from total (parental) DU145 and LNCaP cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. .. PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic or bisulfite treated DNA.

    other:

    Article Title: Comprehensive Evaluation of Commercial Bisulfite-Based DNA Methylation Kits and Development of an Alternative Protocol With Improved Conversion Performance
    Article Snippet: – 5ʹ-formylC was well converted with the EpiTect Bisulfite Kit when conducting two successive sulfonation cycles with an acceptable compromise between conversion of 5ʹ-formylC and presence of unconverted 5ʹ-methylC and 5ʹ-hydroxymethylC.

    Article Title: Comprehensive Evaluation of Commercial Bisulfite-Based DNA Methylation Kits and Development of an Alternative Protocol With Improved Conversion Performance
    Article Snippet: The best results were obtained with the EpiTect Bisulfite Kit with two rounds of sulfonation, however, harming DNA integrity and time-/cost-efficiency.

    Methylation:

    Article Title: Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells
    Article Snippet: .. Methylation Specific polymerase chain reaction (MSP-PCR) A total of 1 μg of DNA extracted from total (parental) DU145 and LNCaP cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. .. PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic or bisulfite treated DNA.

    Article Title: Comprehensive Evaluation of Commercial Bisulfite-Based DNA Methylation Kits and Development of an Alternative Protocol With Improved Conversion Performance
    Article Snippet: .. Results The following bisulfite conversion kits were chosen for testing: EZ DNA Methylation Kit, EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Lightning Kit (all from Zymo Research); EpiTect Bisulfite Kit, EpiTect Fast Bisulfite Kit (both from Qiagen); innuCONVERT Bisulfite Basic Kit (Analytik Jena, Jena, Germany); TrueMethyl Seq Kit (Cambridge Epigenetics, CEGX, Essex, UK); and the Epi proColon 2.0 Kit (Epigenomics AG, Berlin, Germany). .. The tested kits were selected based on the frequency of usage in the international epigenetics community (the most commonly used kits offered by the two worldwide leading companies in this field are sold by Qiagen and Zymo Research).

    DNA Methylation Assay:

    Article Title: Comprehensive Evaluation of Commercial Bisulfite-Based DNA Methylation Kits and Development of an Alternative Protocol With Improved Conversion Performance
    Article Snippet: .. Results The following bisulfite conversion kits were chosen for testing: EZ DNA Methylation Kit, EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Lightning Kit (all from Zymo Research); EpiTect Bisulfite Kit, EpiTect Fast Bisulfite Kit (both from Qiagen); innuCONVERT Bisulfite Basic Kit (Analytik Jena, Jena, Germany); TrueMethyl Seq Kit (Cambridge Epigenetics, CEGX, Essex, UK); and the Epi proColon 2.0 Kit (Epigenomics AG, Berlin, Germany). .. The tested kits were selected based on the frequency of usage in the international epigenetics community (the most commonly used kits offered by the two worldwide leading companies in this field are sold by Qiagen and Zymo Research).

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    Qiagen bisulfite treated dna
    Gene expression level of MGMT is associated with promoter <t>DNA</t> methylation status . MGMT gene expression in methylated (blue box plots) and unmethylated (red box plots) tissue samples analyzed by two different primer/probe sets (Assay 1; Hs00172470_m1 and assay 2; Hs01037698_m1). ( A ) Methylation status based on results from qMSP. ( B ) Methylation status based on results from pyrosequencing. Abbreviation: qMSP, quantitative methylation specific <t>PCR</t>
    Bisulfite Treated Dna, supplied by Qiagen, used in various techniques. Bioz Stars score: 94/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gene expression level of MGMT is associated with promoter DNA methylation status . MGMT gene expression in methylated (blue box plots) and unmethylated (red box plots) tissue samples analyzed by two different primer/probe sets (Assay 1; Hs00172470_m1 and assay 2; Hs01037698_m1). ( A ) Methylation status based on results from qMSP. ( B ) Methylation status based on results from pyrosequencing. Abbreviation: qMSP, quantitative methylation specific PCR

    Journal: Journal of Translational Medicine

    Article Title: MGMT promoter methylation in gliomas-assessment by pyrosequencing and quantitative methylation-specific PCR

    doi: 10.1186/1479-5876-10-36

    Figure Lengend Snippet: Gene expression level of MGMT is associated with promoter DNA methylation status . MGMT gene expression in methylated (blue box plots) and unmethylated (red box plots) tissue samples analyzed by two different primer/probe sets (Assay 1; Hs00172470_m1 and assay 2; Hs01037698_m1). ( A ) Methylation status based on results from qMSP. ( B ) Methylation status based on results from pyrosequencing. Abbreviation: qMSP, quantitative methylation specific PCR

    Article Snippet: Bisulfite treated DNA was amplified in a PCR reaction using primers from the PyroMark Q96 CpG MGMT kit (part number 972032, Qiagen).

    Techniques: Expressing, DNA Methylation Assay, Methylation, Polymerase Chain Reaction

    Primer selection and analytical performance of methylation-specific PCR. A) Schematic illustration of the human INS gene promoter region showing the position of the nine CpG sites. Solid arrows represent the bisulfite-specific primers (BSPs) that amplify both methylated and unmethylated DNA. Dashed arrows represent methylation-specific primers (MSPs) that amplify unmethylated DNA only. B) Unmethylated plasmid was serially diluted after bisulfite conversion and analyzed by qMSP using selected primer sets. Agarose gel electrophoresis of MSP reactions showing the size of the PCR products. C) Graphs of real-time SYBR Green PCR data showing linearity of C q versus log copy number of unmethylated plasmid (averages and standard deviation (SD)) from 5 to 10 6 copies.

    Journal: PLoS ONE

    Article Title: Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

    doi: 10.1371/journal.pone.0094591

    Figure Lengend Snippet: Primer selection and analytical performance of methylation-specific PCR. A) Schematic illustration of the human INS gene promoter region showing the position of the nine CpG sites. Solid arrows represent the bisulfite-specific primers (BSPs) that amplify both methylated and unmethylated DNA. Dashed arrows represent methylation-specific primers (MSPs) that amplify unmethylated DNA only. B) Unmethylated plasmid was serially diluted after bisulfite conversion and analyzed by qMSP using selected primer sets. Agarose gel electrophoresis of MSP reactions showing the size of the PCR products. C) Graphs of real-time SYBR Green PCR data showing linearity of C q versus log copy number of unmethylated plasmid (averages and standard deviation (SD)) from 5 to 10 6 copies.

    Article Snippet: In First-Step PCR, each reaction contained 20–30 ng of bisulfite-treated DNA, 12.5 μl QuantiTect SYBR Green PCR (QIAGEN, Valencia, CA) and 500 nM each forward and reverse primer ( ) in a total volume of 25 μl.

    Techniques: Selection, Methylation, Polymerase Chain Reaction, Plasmid Preparation, Agarose Gel Electrophoresis, SYBR Green Assay, Standard Deviation

    Beta cell specificity of MSP. Serial dilutions of the bisulfite-converted gDNA obtained from human islets, blood, spleen and colon were used as a template for nested PCR using either BSP (A) or MSP (B) in the first-step reaction. The products were used as a template for the second-step MSP reaction. The data display the mean ± SEM of the Relative Unmethylation Ratio (RUR). The cloned INS promoter was used for normalization and standardization of the results. Statistically significant differences at each DNA concentration between islets and other tissues were calculated using two way ANOVA and the significance level indicated by asterisks (****, p

    Journal: PLoS ONE

    Article Title: Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

    doi: 10.1371/journal.pone.0094591

    Figure Lengend Snippet: Beta cell specificity of MSP. Serial dilutions of the bisulfite-converted gDNA obtained from human islets, blood, spleen and colon were used as a template for nested PCR using either BSP (A) or MSP (B) in the first-step reaction. The products were used as a template for the second-step MSP reaction. The data display the mean ± SEM of the Relative Unmethylation Ratio (RUR). The cloned INS promoter was used for normalization and standardization of the results. Statistically significant differences at each DNA concentration between islets and other tissues were calculated using two way ANOVA and the significance level indicated by asterisks (****, p

    Article Snippet: In First-Step PCR, each reaction contained 20–30 ng of bisulfite-treated DNA, 12.5 μl QuantiTect SYBR Green PCR (QIAGEN, Valencia, CA) and 500 nM each forward and reverse primer ( ) in a total volume of 25 μl.

    Techniques: Nested PCR, Clone Assay, Concentration Assay