methylation assay pyroassay  (Pyrosequencing Inc)

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

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