pyrosequencing methylation analysis  (Pyrosequencing Inc)

Journal: European Heart Journal Open

Article Title: Ischaemic preconditioning regulates cardiac transcriptome via DNA methylation conferring cardio-protection from ischaemic reperfusion injury

doi: 10.1093/ehjopen/oeaf124

Figure Lengend Snippet: Ischaemic preconditioning effects on DNA methyltransferase activity and DNA methylation at gene promoters. ( A ) DNA methyltransferase activity of ischaemic preconditioning vs. non-ischaemic preconditioning at T1 ( n = 10 per group. ( B ) RT-PCR data for mRNA levels of the CCAAT enhancer binding protein delta (Cebpd) gene ( n = 10 per group). ( C ) Schematic diagram showing Cebpd genomic location, CpG island, and regions amplified with primers designed within CpG island and gene promoter region ( D ) MethylMiner quantitative PCR data showing relative promoter methylation for Regions 1 and 2 of the Cebpd gene ( n = 6 per group). ( E ) RT-PCR data for mRNA levels of the NFKB inhibitor alpha (Nfkbia ) gene ( n = 10 per group). ( F ) Schematic diagram showing Nfkbia genomic location, CpG island, and regions amplified with primers designed within CpG island and gene promoter region ( G ) MethylMiner quantitative PCR data showing relative promoter methylation for Regions 1 and 2 of the Nfkbia gene ( n = 6 per group). ( H ) RT-PCR data for mRNA levels of the Growth arrest and DNA damage inducible beta (Gadd45b) gene ( n = 10 per group). ( I ) Schematic diagram showing Gadd45b genomic location, CpG island, and regions amplified with primers designed within CpG island and gene promoter region ( J ) MethylMiner quantitative PCR data showing relative promoter methylation for Regions 1 and 2 of the Gadd45b gene ( n = 6 per group). ( K ) RT-PCR data for mRNA levels of the Jun proto-oncogene ( Jun ) gene ( n = 10 per group). ( L ) Schematic diagram showing Jun genomic location, CpG island, and regions amplified with primers designed within CpG island and gene promoter region ( M ) MethylMiner quantitative PCR data showing relative promoter methylation for Regions 1 and 2 of the Jun gene ( n = 6 per group). ( N ) RT-PCR data for mRNA levels of the Apolipoprotein L domain containing 1 ( Aplod1 ) gene ( n = 10 per group). ( O ) Schematic diagram showing Aplod1 genomic location, CpG island, and regions amplified with primers designed within CpG island and gene promoter region ( P ) MethylMiner quantitative PCR data showing relative promoter methylation for Regions 1 and 2 of the Aplod1 gene ( n = 6 per group). Data are shown as mean ± SEM. P -value was determined using an unpaired two-tailed t -test.

Article Snippet: DNA methylation analysis was performed using a MethylMiner Enrichment Kit (Invitrogen, CA, USA).

Techniques: Activity Assay, DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Amplification, Real-time Polymerase Chain Reaction, Methylation, Two Tailed Test

Journal: European Heart Journal Open

Article Title: Ischaemic preconditioning regulates cardiac transcriptome via DNA methylation conferring cardio-protection from ischaemic reperfusion injury

doi: 10.1093/ehjopen/oeaf124

Figure Lengend Snippet: Ischaemic preconditioning effects on DNA methyltransferase activity and DNA methylation at gene promoters in presence of prolonged ischaemia. ( A ) DNA methyltransferase activity of ischaemic preconditioning vs. non-ischaemic preconditioning at T2 ( n = 10 per group. ( B ) RT-PCR data for mRNA levels of the Transmembrane protein 200C ( Tmem200c ) gene ( n = 10 per group). ( C ) Schematic diagram showing Tmem200c genomic location and CpG island (green line). Red lines indicate the CpG rich regions amplified with specific primers designed within CpG island and gene promoter ( D ) MethylMiner quantitative PCR data showing relative promoter methylation for Regions 1 and 2 of the Tmem200c gene ( n = 6 per group). ( E ) RT-PCR data for mRNA levels of the Heat shock protein family H member 1 (Hsph1 ) gene ( n = 10 per group). ( F ) Schematic diagram showing Hsph1 genomic location and CpG island (green line). Red lines indicate the CpG rich regions amplified with specific primers designed within CpG island and gene promoter ( G ) MethylMiner quantitative PCR data showing relative promoter methylation for Regions 1 and 2 of the Hsph1 gene ( n = 6 per group). ( H ) RT-PCR data for mRNA levels of the Fibroblast growth factor receptor 4 (Fgfr4) gene ( n = 10 per group). ( I ) Schematic diagram showing Fgfr4 genomic location and CpG island (green line). Red lines indicate the CpG rich regions amplified with specific primers designed within CpG island and gene promoter. ( J ) MethylMiner quantitative PCR data showing relative promoter methylation for Regions 1 and 2 of the Fgfr4 gene ( n = 6 per group). ( K ) RT-PCR data for mRNA levels of the Serine/Threonine kinase 32C ( Stk32c ) gene ( n = 10 per group). ( L ) Schematic diagram showing Stk32 genomic location and CpG island (line underneath). Red lines indicate the CpG rich regions amplified with specific primers designed within CpG island and gene promoter ( M ) MethylMiner quantitative PCR data showing relative promoter methylation for Regions 1 and 2 of the Stk32 gene ( n = 6 per group). ( N ) RT-PCR data for mRNA levels of the Poly (ADP-Ribose) polymerase family member 14 ( Parp14 ) gene ( n = 10 per group). ( O ) Schematic diagram showing Parp14 genomic location and CpG island (green line). Red lines indicate the CpG rich regions amplified with specific primers designed within CpG island and gene promoter ( P ) MethylMiner quantitative PCR data showing relative promoter methylation for Regions 1 and 2 of the Parp14 gene ( n = 6 per group). Data are shown as mean ± SEM. P -value was determined using an unpaired two-tailed t -test.

Article Snippet: DNA methylation analysis was performed using a MethylMiner Enrichment Kit (Invitrogen, CA, USA).

Techniques: Activity Assay, DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Methylation, Two Tailed Test