bisulfite-pyrosequencing primer  (Pyrosequencing Inc)

Journal: Clinical Epigenetics

Article Title: Promoter methylation changes in ALOX12 and AIRE1 : novel epigenetic markers for atherosclerosis

doi: 10.1186/s13148-020-00846-0

Figure Lengend Snippet: Promoter CpG islands and regions for the pyrosequencing of the five target genes identified after methylation-specific PCR ( a ) and methylation levels of the genes in plaques (P) and non-plaque (NP) intima of common carotid arteries in 20 cadavers ( b ). Correlation between promoter methylation and expression of AIRE1 ( c ) and ALOX12 ( d ) in 18 carotid endarterectomy plaques. Open bar, exon 1 region; closed bar, the regions targeted for bisulfite pyrosequencing; open bar in the middle of closed bar; sequencing region, arrow; transcriptional start site of each gene, * p < 0.05 on paired t test

Article Snippet: We used bisulfite pyrosequencing primer sets for AIRE1 (AIRE1-F; biotin-5′-GGGTAGTTTTTGTTAGGGTTTTGA-3′ and AIRE1-R; 5′-TCACCCACCAAAACAACTACCTTA-3′ for PCR and AIRE1-S; 5′-TCAAAAAACTCTCACTTCC-3′ for bisulfite pyrosequencing) and NEOT1 (NEOT1-F; biotin-5′-GTGGGGGGTTTAATTGTAAGAAGT-3′ and NETO1-R; 5′-TCTCTACCTCCCACCCCTTCTTT-3′ for PCR and NETO1-S; 5′-CCCCTTCTTTTCCAT-3′ for bisulfite pyrosequencing) (Fig. a).

Techniques: Methylation, Expressing, Sequencing

Journal: Clinical Epigenetics

Article Title: Promoter methylation changes in ALOX12 and AIRE1 : novel epigenetic markers for atherosclerosis

doi: 10.1186/s13148-020-00846-0

Figure Lengend Snippet: Immunofluorescence staining for AIRE1 and infiltrated CD4(+)-T cells and CD14(+)-monocytes in atherosclerotic plaque and non-plaque intima of common carotid artery of a 54-year-old man. White arrow, cells co-stained with CD14 and AIRE1 antibodies

Article Snippet: We used bisulfite pyrosequencing primer sets for AIRE1 (AIRE1-F; biotin-5′-GGGTAGTTTTTGTTAGGGTTTTGA-3′ and AIRE1-R; 5′-TCACCCACCAAAACAACTACCTTA-3′ for PCR and AIRE1-S; 5′-TCAAAAAACTCTCACTTCC-3′ for bisulfite pyrosequencing) and NEOT1 (NEOT1-F; biotin-5′-GTGGGGGGTTTAATTGTAAGAAGT-3′ and NETO1-R; 5′-TCTCTACCTCCCACCCCTTCTTT-3′ for PCR and NETO1-S; 5′-CCCCTTCTTTTCCAT-3′ for bisulfite pyrosequencing) (Fig. a).

Techniques: Immunofluorescence, Staining

Journal: Clinical Epigenetics

Article Title: Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI , GTF2H4 , and TNXB genes

doi: 10.1186/s13148-019-0608-2

Figure Lengend Snippet: Bisulfite pyrosequencing of candidate gene SKI (cg18934822) validates direction of methylation change identified in 450k array. a – c Univariate analysis of SKI (cg18934822) in Illumina 450k BeadChip array. a Methylation β values for AMD ( n = 25) compared to normal ( n = 19) donor RPE cells after normalization, in addition to analysis of sex-stratified results for cg18934822 ( SKI ). Significantly reduced methylation levels are observed in AMD ( p < 0.0001), as well as in both AMD male ( n = 15) ( b ) and AMD female ( n = 10) ( c ) compared to normal male ( n = 12) ( p = 0.0063) and normal female ( n = 7) ( p = 0.0031) human RPE donor cells respectively. d – f Bisulfite pyrosequencing of candidate gene SKI (cg18934822) in combined technical and independent sample replications. d Reduced methylation of cg18934822 ( SKI ) is identified in AMD ( n = 30) compared to normal donor RPE cells ( n = 25) ( p = 0.0309). e Reduced methylation of cg18934822 ( SKI ) is identified in AMD AMD male ( n = 20) compared to normal male ( n = 18) ( p = 0.1223) and f AMD female ( n = 10) compared to normal female donor samples ( n = 7) ( p = 0.1002). (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). All statistical analysis was performed using the Mann-Whitney U test

Article Snippet: Bisulfite Pyrosequencing primer sequences.

Techniques: Methylation, MANN-WHITNEY

Journal: Clinical Epigenetics

Article Title: Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI , GTF2H4 , and TNXB genes

doi: 10.1186/s13148-019-0608-2

Figure Lengend Snippet: Bisulfite pyrosequencing of candidate gene GTF2H4 (cg22508626) validates direction of methylation change identified in 450k array. a–c Univariate analysis of GTF2H4 cg22508626 in Illumina 450k BeadChip array. a Methylation β values for AMD ( n = 25) compared to normal RPE donor cells ( n = 19) after normalization, in addition to analysis of sex-stratified results for cg22508626 ( GTF2H4 ). Significantly increased methylation levels are observed in AMD compared to normal ( p = 0.0003), in addition to both b AMD male ( n = 15) and c AMD female ( n = 10) compared to normal male ( n = 12) ( p = 0.0074) and normal female ( n = 7) ( p = 0.0136) human donor samples respectively. d – f Bisulfite pyrosequencing of candidate gene GTF2H4 (cg22508626) in combined technical and independent sample replications. d Mean methylation difference observed in cg22508626 ( GTF2H4 ) in AMD ( n = 29) compared to normal donor samples ( n = 24) does not reach statistical significance ( p = 0.1263). Sex-stratified analysis reveals no significant methylation differences in e AMD male ( n = 18) versus normal male ( n = 18) donor samples. Significantly increased methylation is observed in f AMD female ( n = 11) versus normal male ( n = 6) ( p = 0.0292). (* p ≤ 0.05)(** p ≤ 0.01)(*** p ≤ 0.001)(**** p ≤ 0.0001). All statistical analysis was performed using the Mann-Whitney U test

Article Snippet: Bisulfite Pyrosequencing primer sequences.

Techniques: Methylation, MANN-WHITNEY

Journal: Clinical Epigenetics

Article Title: Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI , GTF2H4 , and TNXB genes

doi: 10.1186/s13148-019-0608-2

Figure Lengend Snippet: Bisulfite pyrosequencing of candidate genes: RIC3 (cg01560972), FAIM2 (cg18486102), EIF2AK3 (cg26347887), and GRIA4 (cg03243226). a No significant mean methylation difference was observed for cg01560972 ( RIC3 ) in AMD ( n = 30) compared to normal ( n = 25) donor RPE cells (p = 0.9491). b Sex-stratified analysis did not reveal significant differences in AMD male ( n = 18) compared to normal male ( n = 18) ( p = 0.2006). c A significant hypermethylation was observed in AMD female ( n = 12) compared to normal female ( n = 7) human donor samples ( p = 0.0327). d No significant mean methylation difference was observed for cg18486102 ( FAIM2 ) in AMD ( n = 30) compared to normal donor RPE cells ( n = 25) ( p = 0.1672). e Sex-stratified analysis revealed significant differential hypomethylation in males (AMD male n = 19, normal male, n = 18) ( p = 0.0276). No significant mean methylation difference was observed in AMD female ( n = 11) compared to normal female ( n = 7) human donor RPE cells ( p = 0.4362). f No significant methylation difference was observed in cg26347887 ( EIF2AK3 ) in AMD ( n = 28) compared to human donor samples ( n = 23) ( p = 0.4546) g – i . Sex-stratified results did not reveal significant methylation changes in males or females. j No significant mean methylation difference was observed for cg03243226 ( GRIA4 ) in AMD ( n = 30) compared to human donor RPE cells ( n = 25) ( p = 0.2053). k Sex-stratified analysis did not show significant differential hypomethylation in AMD male ( n = 18) compared to normal male ( n = 18) ( p = 0.8197); however, a significant mean methylation difference was observed in AMD female ( n = 12) compared to normal female ( n = 7) ( p = 0.0279) human donor RPE samples ( l ). All statistical analysis was performed using the Mann-Whitney U test (* p ≤ 0.05)

Article Snippet: Bisulfite Pyrosequencing primer sequences.

Techniques: Methylation, MANN-WHITNEY

Journal: Clinical Epigenetics

Article Title: Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI , GTF2H4 , and TNXB genes

doi: 10.1186/s13148-019-0608-2

Figure Lengend Snippet: Global methylation analysis of LINE-1 using bisulfite pyrosequencing of AMD RPE cells compared to normals. Mean methylation of LINE-1 compared to normals using bisulfite pyrosequencing. a LINE-1 analysis did not identify significant differences between AMD donor RPE cells ( n = 29) and normal donor RPE cells ( n = 23) ( p = 0.8788). Sex-stratified analyses revealed no significant methylation differences in AMD male ( n = 17) compared to normal male ( n = 18) ( p = 0.7730) ( b ) or AMD female ( n = 12) compared to normal female human RPE cells ( n = 5) ( p = 0.8361) ( c ). All statistical analysis was performed using the Mann-Whitney U test

Article Snippet: Bisulfite Pyrosequencing primer sequences.

Techniques: Methylation, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection

doi: 10.1371/journal.pone.0192722

Figure Lengend Snippet: Mixtures of bisulfite-converted and unconverted NCAs were subjected to bisulfite pyrosequencing using sequencing primers A9515 or hND1. Each bar represents mean±SD of three independent analyses.

Article Snippet: To our surprise, the bisulfite pyrosequencing assay using the hND1 primer was extremely sensitive to the presence of very small amounts of unconverted NCA, and the impact of unconverted NCA remarkably differed between the CpG sites.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection

doi: 10.1371/journal.pone.0192722

Figure Lengend Snippet: Ratios of brCs were determined by bisulfite pyrosequencing (Mean±SD, triplicated assays). (A) The A9515 sequencing primer, which was highly selective to bisulfite-converted DNA, interrogated three CpG sites (CpG #3–5) whereas non-selective sequencing primer hND1 interrogated all these CpG sites plus two additional CpG sites (CpG #1 and 2). (B) Positive control assay was performed using in vitro partially methylated NCAs templates. High CpG methylation levels at three CpG sites (CpG #3–5) were detected using A9515 sequencing primer (CpG sites #1 and #2 were out of the assay coverage using this sequencing primer). hND1 sequencing primer detected high CpG methylation at all five CpG sites (CpG #1–5).

Article Snippet: To our surprise, the bisulfite pyrosequencing assay using the hND1 primer was extremely sensitive to the presence of very small amounts of unconverted NCA, and the impact of unconverted NCA remarkably differed between the CpG sites.

Techniques: Sequencing, Positive Control Assay, In Vitro, Methylation, CpG Methylation Assay