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Comparison of DNA methylation levels assessed using Illumina HM450K array and pyrosequencing assays <t> (PyroAssays) </t>

Comparison of DNA methylation levels assessed using Illumina HM450K array and pyrosequencing assays <t> (PyroAssays) </t>

Pyroassays, supplied by Pyrosequencing Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pyroassays/product/Pyrosequencing Inc
Average 90 stars, based on 1 article reviews

pyroassays - by Bioz Stars, 2026-04

90/100 stars

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1) Product Images from "Discrimination between human populations using a small number of differentially methylated CpG sites: a preliminary study using lymphoblastoid cell lines and peripheral blood samples of European and Chinese origin"

Article Title: Discrimination between human populations using a small number of differentially methylated CpG sites: a preliminary study using lymphoblastoid cell lines and peripheral blood samples of European and Chinese origin

Journal: BMC Genomics

doi: 10.1186/s12864-020-07092-x

Figure Legend Snippet: Comparison of DNA methylation levels assessed using Illumina HM450K array and pyrosequencing assays (PyroAssays)

Techniques Used: DNA Methylation Assay, Methylation

Results of the technical validation of eight PyroAssays. Twenty B-lymphocyte cell lines (10 from each population) were tested. The originally selected candidate pop-CpGs targeted in each PyroAssay are marked with *. Green – CEU population; blue – CHB population. Dots represent methylation levels in individual samples. Box plots denote mean value (lines inside the boxes) and standard deviation. Statistically significant ( p < 0.05) population differences in the methylation level are marked in red

Figure Legend Snippet: Results of the technical validation of eight PyroAssays. Twenty B-lymphocyte cell lines (10 from each population) were tested. The originally selected candidate pop-CpGs targeted in each PyroAssay are marked with *. Green – CEU population; blue – CHB population. Dots represent methylation levels in individual samples. Box plots denote mean value (lines inside the boxes) and standard deviation. Statistically significant ( p < 0.05) population differences in the methylation level are marked in red

Techniques Used: Methylation, Standard Deviation

Figure Legend Snippet: Validation of eight PyroAssays performed in the independent set of B-lymphocyte cell lines

Techniques Used:

Correlation matrix showing the results of Pearson correlation analysis. Analysis was performed using data from PyroAssays performed in 20 B-lymphocyte cell lines ( n = 10 from CEU, n = 10 from CHB population). Pearson correlation coefficient values and directions are marked with different colors; positive correlation (from white to red on the color scale); negative correlation (from white to blue) (see color-bar next to the matrix)

Figure Legend Snippet: Correlation matrix showing the results of Pearson correlation analysis. Analysis was performed using data from PyroAssays performed in 20 B-lymphocyte cell lines ( n = 10 from CEU, n = 10 from CHB population). Pearson correlation coefficient values and directions are marked with different colors; positive correlation (from white to red on the color scale); negative correlation (from white to blue) (see color-bar next to the matrix)

Techniques Used:

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Journal: BMC Genomics

doi: 10.1186/s12864-020-07092-x

Figure Lengend Snippet: Comparison of DNA methylation levels assessed using Illumina HM450K array and pyrosequencing assays (PyroAssays)

Article Snippet: Pyrosequencing assays (further referred as PyroAssays) were designed to validate candidate pop-CpG sites preselected in HM450K array experiment for which effective PyroAssays could be designed (Assay score in PyroMark Assay Design Software ≥75, no CpGs under PyroAssay primers); in some cases, PyroAssays covered additional CpGs located in the close proximity (less than 25 bp upstream or downstream) of the selected candidate pop-CpGs (see Table in section).

Techniques: DNA Methylation Assay, Methylation

Journal: BMC Genomics

doi: 10.1186/s12864-020-07092-x

Figure Lengend Snippet: Results of the technical validation of eight PyroAssays. Twenty B-lymphocyte cell lines (10 from each population) were tested. The originally selected candidate pop-CpGs targeted in each PyroAssay are marked with *. Green – CEU population; blue – CHB population. Dots represent methylation levels in individual samples. Box plots denote mean value (lines inside the boxes) and standard deviation. Statistically significant ( p < 0.05) population differences in the methylation level are marked in red

Techniques: Methylation, Standard Deviation

Journal: BMC Genomics

doi: 10.1186/s12864-020-07092-x

Figure Lengend Snippet: Validation of eight PyroAssays performed in the independent set of B-lymphocyte cell lines

Techniques:

Journal: BMC Genomics

doi: 10.1186/s12864-020-07092-x

Figure Lengend Snippet: Correlation matrix showing the results of Pearson correlation analysis. Analysis was performed using data from PyroAssays performed in 20 B-lymphocyte cell lines ( n = 10 from CEU, n = 10 from CHB population). Pearson correlation coefficient values and directions are marked with different colors; positive correlation (from white to red on the color scale); negative correlation (from white to blue) (see color-bar next to the matrix)

Techniques:

Top ranking promoter regions included imprint regulator gene ZFP57 . a Top 5 differentially methylated regions (DMR) at promoters, sorted by combined RnBeads rank (smallest to largest) as for Fig. d above, except combining values across all the CpG sites in the DMR as detailed in the “ ” section. Abbreviations as above except # probes, number of probes on EPIC array included in DMR. b Top: genome browser tracks showing the region around the DMR upstream of ZFP57 , genomic coordinates in hg19 human genome release, and scale as shown. EPIC array probes showing differential methylation (blue, gain; red, loss) are indicated, with size indicating the magnitude of change. The start of the ZFP57 gene and the position of the pyrosequencing assay (Pyro) are also shown. Δ β , mean difference in β value between placebo and FA-treated groups; maximum gain and loss also shown (+ 0.09 β = 9% methylation). Bottom: Loess plot of β values across the region, with CpG identification numbers from array below; those forming the DMR defined by RnBeads are indicated, as well as sites analyzed by pyroassay. Each dot represents β value in an individual sample, with lines representing smoothed averages; color code is indicated at left. c Results of pyroassay covering the six sites indicated in b . Sample groups: cord blood DNA from placebo ( n = 45) and FA-treated ( n = 41). Mean, average of the individual means in that group; Max., largest of the mean methylation values in that group; Min, lowest mean in group; SD, standard deviation for the means; Change, difference in % methylation seen between groups; P, probability (Student’s t test)

Journal: Clinical Epigenetics

Article Title: A randomized controlled trial of folic acid intervention in pregnancy highlights a putative methylation-regulated control element at ZFP57

doi: 10.1186/s13148-019-0618-0

Figure Lengend Snippet: Top ranking promoter regions included imprint regulator gene ZFP57 . a Top 5 differentially methylated regions (DMR) at promoters, sorted by combined RnBeads rank (smallest to largest) as for Fig. d above, except combining values across all the CpG sites in the DMR as detailed in the “ ” section. Abbreviations as above except # probes, number of probes on EPIC array included in DMR. b Top: genome browser tracks showing the region around the DMR upstream of ZFP57 , genomic coordinates in hg19 human genome release, and scale as shown. EPIC array probes showing differential methylation (blue, gain; red, loss) are indicated, with size indicating the magnitude of change. The start of the ZFP57 gene and the position of the pyrosequencing assay (Pyro) are also shown. Δ β , mean difference in β value between placebo and FA-treated groups; maximum gain and loss also shown (+ 0.09 β = 9% methylation). Bottom: Loess plot of β values across the region, with CpG identification numbers from array below; those forming the DMR defined by RnBeads are indicated, as well as sites analyzed by pyroassay. Each dot represents β value in an individual sample, with lines representing smoothed averages; color code is indicated at left. c Results of pyroassay covering the six sites indicated in b . Sample groups: cord blood DNA from placebo ( n = 45) and FA-treated ( n = 41). Mean, average of the individual means in that group; Max., largest of the mean methylation values in that group; Min, lowest mean in group; SD, standard deviation for the means; Change, difference in % methylation seen between groups; P, probability (Student’s t test)

Article Snippet: To confirm these results using a second method, we designed a pyrosequencing methylation assay (pyroassay) to cover some of these CpG sites, as shown in Fig. b.

Techniques: Methylation, Pyrosequencing Assay, Standard Deviation

ZFP57 upstream region is a methylation-dependent regulator of transcription at this locus. a Schematic as in Fig. above but showing difference in methylation (Δ β ) between HCT116 WT cells vs HCT116 DKO cells. The intron/exon structure and positions of the forward (FW) and reverse (RV) primers for RT-(q)PCR on the ZFP57 gene are also shown. b Methylation levels at individual CpG covered by the pyrosequencing assay in WT (HCT116) and knockout (DKO) cells. Values are shown as mean +/− SD for each site: * p < 0.05; ** p < 0.01; *** p < 0.001. c RT-PCR showing upregulation using the primers indicated in a , key as above. CTRL, positive control (human reference total RNA); NTC, negative control (no template control); 100 bp, size standards ladder; ACTB , β-actin loading control. d Confirmation of upregulation by RT-qPCR using the same primers, values normalized to HPRT ; FC, fold change. e Methylation levels using pyroassay as in B but in 5-aza-dC treated SH-SY5Y cells (5-aza-dC), as compared to untreated (UT). f RT-PCR for 5-aza-dC treated cells from e . g RT-qPCR confirmation of ZFP57 upregulation in 5-aza-dC-treated SH-SY5Y cells

Journal: Clinical Epigenetics

Article Title: A randomized controlled trial of folic acid intervention in pregnancy highlights a putative methylation-regulated control element at ZFP57

doi: 10.1186/s13148-019-0618-0

Figure Lengend Snippet: ZFP57 upstream region is a methylation-dependent regulator of transcription at this locus. a Schematic as in Fig. above but showing difference in methylation (Δ β ) between HCT116 WT cells vs HCT116 DKO cells. The intron/exon structure and positions of the forward (FW) and reverse (RV) primers for RT-(q)PCR on the ZFP57 gene are also shown. b Methylation levels at individual CpG covered by the pyrosequencing assay in WT (HCT116) and knockout (DKO) cells. Values are shown as mean +/− SD for each site: * p < 0.05; ** p < 0.01; *** p < 0.001. c RT-PCR showing upregulation using the primers indicated in a , key as above. CTRL, positive control (human reference total RNA); NTC, negative control (no template control); 100 bp, size standards ladder; ACTB , β-actin loading control. d Confirmation of upregulation by RT-qPCR using the same primers, values normalized to HPRT ; FC, fold change. e Methylation levels using pyroassay as in B but in 5-aza-dC treated SH-SY5Y cells (5-aza-dC), as compared to untreated (UT). f RT-PCR for 5-aza-dC treated cells from e . g RT-qPCR confirmation of ZFP57 upregulation in 5-aza-dC-treated SH-SY5Y cells

Article Snippet: To confirm these results using a second method, we designed a pyrosequencing methylation assay (pyroassay) to cover some of these CpG sites, as shown in Fig. b.

Techniques: Methylation, Pyrosequencing Assay, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Quantitative RT-PCR

Loss of methylation at the protocadherin γ ( PCDHG ) cluster of neuroepithelial identity genes. a Structure of the PCDHG cluster showing the 5′ variable exons (A, B and C classes) which are spliced to the 3′ constant exons (right). The top track (amber) shows absolute β values in the WT fibroblast cells from the 450K array, which range from 1(fully methylated) to zero (unmethylated). Only the sites showing significant differences from WT (FDR < 0.05) in each cell line are shown in the three tracks below, with decreases in red representing loss of methylation, and gains in blue. The size of the bar is proportional to the magnitude of change: maxima and minima are indicated on the scales at left. The locations of CpG islands (CGI) are also shown. Pyroassay locations are boxed. b Median β values for all variable exons. Significant differences (Mann–Whitney U ) are indicated: * p < 0.05; ** p < 0.1; n.s., not significant. c Methylation at each exon in WT and d16 cells obtained by taking the median of the absolute β value for all probes at that exon. The variable class C exons are underlined. d Average methylation values in WT and KD cells obtained from a pyrosequencing assay (pyroassay) designed to cover CpGs in the A2 exon. Bars represent SEM; *** p < 0.001, t -test. e Methylation at the C4 variable exon by pyroassay, shown as a control

Journal: Epigenetics & Chromatin

Article Title: Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression

doi: 10.1186/s13072-018-0182-4

Figure Lengend Snippet: Loss of methylation at the protocadherin γ ( PCDHG ) cluster of neuroepithelial identity genes. a Structure of the PCDHG cluster showing the 5′ variable exons (A, B and C classes) which are spliced to the 3′ constant exons (right). The top track (amber) shows absolute β values in the WT fibroblast cells from the 450K array, which range from 1(fully methylated) to zero (unmethylated). Only the sites showing significant differences from WT (FDR < 0.05) in each cell line are shown in the three tracks below, with decreases in red representing loss of methylation, and gains in blue. The size of the bar is proportional to the magnitude of change: maxima and minima are indicated on the scales at left. The locations of CpG islands (CGI) are also shown. Pyroassay locations are boxed. b Median β values for all variable exons. Significant differences (Mann–Whitney U ) are indicated: * p < 0.05; ** p < 0.1; n.s., not significant. c Methylation at each exon in WT and d16 cells obtained by taking the median of the absolute β value for all probes at that exon. The variable class C exons are underlined. d Average methylation values in WT and KD cells obtained from a pyrosequencing assay (pyroassay) designed to cover CpGs in the A2 exon. Bars represent SEM; *** p < 0.001, t -test. e Methylation at the C4 variable exon by pyroassay, shown as a control

Article Snippet: Key to tracks as before; pyroassay locations ( UGT1A1 and UGT1A4 ) are boxed. b Median β values for all first exons: though medians are higher in KD lines these failed to reach statistical significance. c Median absolute β values at individual first exons in WT and d16 cells. d Average methylation values in WT and KD cells obtained from a pyrosequencing assay (pyroassay) designed to cover CpGs in the A4 exon. e Methylation by pyroassay at the A1 exon, shown as a control

Techniques: Methylation, MANN-WHITNEY, Pyrosequencing Assay, Control

Gains in methylation on the X chromosome. a Sites reliably showing gain in methylation and located in promoters were analysed to identify those common to all three KD lines ( n = 201). Some of these sites showing the greatest change in methylation were located on the X chromosome including MAGEA12 and GABRQ . b Schematic showing the locations of the two genes adjacent to each other on X in a region showing gain in methylation. Tracks indicate the locations of all 450K probes and CGI; the positions of the pyroassays are also indicated; the scale bar pertains to the bottom part of the schematic; ∆ β , change in beta value. c Methylation as determined by pyroassay at the two genes indicated in a , b . d Clonal analysis of GABRQ in WT and d8. Filled circles represent methylated sites, open circles unmethylated. The CpG which were also analysed by the pyroassay (pyro) and the 450K array (asterisk) are indicated

Journal: Epigenetics & Chromatin

Article Title: Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression

doi: 10.1186/s13072-018-0182-4

Figure Lengend Snippet: Gains in methylation on the X chromosome. a Sites reliably showing gain in methylation and located in promoters were analysed to identify those common to all three KD lines ( n = 201). Some of these sites showing the greatest change in methylation were located on the X chromosome including MAGEA12 and GABRQ . b Schematic showing the locations of the two genes adjacent to each other on X in a region showing gain in methylation. Tracks indicate the locations of all 450K probes and CGI; the positions of the pyroassays are also indicated; the scale bar pertains to the bottom part of the schematic; ∆ β , change in beta value. c Methylation as determined by pyroassay at the two genes indicated in a , b . d Clonal analysis of GABRQ in WT and d8. Filled circles represent methylated sites, open circles unmethylated. The CpG which were also analysed by the pyroassay (pyro) and the 450K array (asterisk) are indicated

Techniques: Methylation

Methylation loss is concentrated at regions normally repressed by polycomb. a Distribution of probes showing significant loss per chromatin state—numbers of probes are shown at left, chromatin states below: tcn, transcription; heterochrom/Lo, heterochromatin or low signal; repetitive, repeat DNA. b Region around the LEP gene: tracks as before, with the addition of data from cells treated with siRNA for 72 h (top). A track showing ChromHMM chromatin states from NHLF foetal lung fibroblasts is shown at bottom: grey, polycomb-repressed; green, transcriptionally active (full colour key at top right). c DNMT1 mRNA levels by qPCR following treatment with siRNA (+) for 72 h compared with scrambled control (Scr). ACTB is shown as a control; ladder as above. d Median β values for all regions (WT) compared to medians for polycomb-repressed regions (Polycomb), or all other regions (Other) in the cell lines indicated at top; remeth, remethylated. e Numbers of probes showing loss and gain in methylation in hTERT cells following treatment with siRNA for 72 h compared with the shRNA lines (averaged); #, number. f mRNA levels for the indicated genes in shRNA lines treated with the EzH2 inhibitor DZNeP; UNT, untreated; bars represent SEM, experiment carried out in duplicate. g Median β values for all variable exons at the PCDHG locus (left) and for fat/body mass genes (FBM, right): compare d16 shRNA lines with cells treated with siRNA. h DNMT1 mRNA levels in WT cells exposed to siRNA for 48 h, then allowed to recover in normal medium; comparisons were made to a scrambled siRNA negative control (Scr). i Methylation levels by pyroassay at the loci indicated during the transient KD and recovery shown in ( h ); timepoints are in days. All loci showed significant loss of methylation: LEP and SNRPN showed no significant gain versus lowest methylation level, while PCDHGA2 showed no significant gain between d22 and d36

Journal: Epigenetics & Chromatin

Article Title: Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression

doi: 10.1186/s13072-018-0182-4

Figure Lengend Snippet: Methylation loss is concentrated at regions normally repressed by polycomb. a Distribution of probes showing significant loss per chromatin state—numbers of probes are shown at left, chromatin states below: tcn, transcription; heterochrom/Lo, heterochromatin or low signal; repetitive, repeat DNA. b Region around the LEP gene: tracks as before, with the addition of data from cells treated with siRNA for 72 h (top). A track showing ChromHMM chromatin states from NHLF foetal lung fibroblasts is shown at bottom: grey, polycomb-repressed; green, transcriptionally active (full colour key at top right). c DNMT1 mRNA levels by qPCR following treatment with siRNA (+) for 72 h compared with scrambled control (Scr). ACTB is shown as a control; ladder as above. d Median β values for all regions (WT) compared to medians for polycomb-repressed regions (Polycomb), or all other regions (Other) in the cell lines indicated at top; remeth, remethylated. e Numbers of probes showing loss and gain in methylation in hTERT cells following treatment with siRNA for 72 h compared with the shRNA lines (averaged); #, number. f mRNA levels for the indicated genes in shRNA lines treated with the EzH2 inhibitor DZNeP; UNT, untreated; bars represent SEM, experiment carried out in duplicate. g Median β values for all variable exons at the PCDHG locus (left) and for fat/body mass genes (FBM, right): compare d16 shRNA lines with cells treated with siRNA. h DNMT1 mRNA levels in WT cells exposed to siRNA for 48 h, then allowed to recover in normal medium; comparisons were made to a scrambled siRNA negative control (Scr). i Methylation levels by pyroassay at the loci indicated during the transient KD and recovery shown in ( h ); timepoints are in days. All loci showed significant loss of methylation: LEP and SNRPN showed no significant gain versus lowest methylation level, while PCDHGA2 showed no significant gain between d22 and d36

Techniques: Methylation, Control, shRNA, Negative Control

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1) Product Images from "Discrimination between human populations using a small number of differentially methylated CpG sites: a preliminary study using lymphoblastoid cell lines and peripheral blood samples of European and Chinese origin"

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