MethPrimer: designing primers for methylation PCRs

Li, Long Cheng; Dahiya, Rajvir

doi:10.1093/bioinformatics/18.11.1427

Cited by 2,309 publications

(1,758 citation statements)

References 20 publications

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“…Successful amplification from the M primers and U primers indicated methylation and unmethylation, respectively. For primer design, a sequence starting 2,000 bp upstream from the transcriptional start site (TSS) of PPARGC1A and Tfam was used in the MethPrimer program (http://www.urogene.org//methprimer/index1.html) 15 to search for regions with potentially methylated CpG sites. The sequence was retrieved from the Database of Transcriptional Start Sites (http://dbtss.hgc.jp/) with the following ID numbers: NM_013261, chromosome: NCBI36: 4: 23402742..23500798, TSS: 23500789 for PPARGC1A, and NM_003201, chromosome: NCBI36: 10: 59815182..59825903, TSS: 59815182 for TFAM.…”

Section: Methodsmentioning

confidence: 99%

Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: Impact of liver methylation of the peroxisome proliferator-activated receptor γ coactivator 1α promoter

et al. 2010

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Insulin resistance (IR) and mitochondrial dysfunction play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). We hypothesized that genetic factors and epigenetic modifications occurring in the liver contribute to the IR phenotype. We specifically examined whether fatty liver and IR are modified by hepatic DNA methylation of the peroxisome proliferator-activated receptor c coactivator 1a (PPARGC1A) and mitochondrial transcription factor A (TFAM) promoters, and also evaluated whether liver mitochondrial DNA (mtDNA) content is associated with NAFLD and IR. We studied liver biopsies obtained from NAFLD patients in a case-control design. After bisulfite treatment of DNA, we used methylation-specific polymerase chain reaction (PCR) to assess the putative methylation of three CpG in the PPARGC1A and TFAM promoters. Liver mtDNA quantification using nuclear DNA (nDNA) as a reference was evaluated by way of realtime PCR. Liver PPARGC1A methylated DNA/unmethylated DNA ratio correlated with plasma fasting insulin levels and homeostasis model assessment of insulin resistance (HOMA-IR); TFAM methylated DNA/unmethylated DNA ratio was inversely correlated with insulin levels. PPARGC1A promoter methylation was inversely correlated with the abundance of liver PPARGC1A messenger RNA. The liver mtDNA/nDNA ratio was significantly higher in control livers compared with NAFLD livers. mtDNA/nDNA ratio was inversely correlated with HOMA-IR, fasting glucose, and insulin and was inversely correlated with PPARGC1A promoter methylation. Conclusion: Our data suggest that the IR phenotype and the liver transcriptional activity of PPARGC1A show a tight interaction, probably through epigenetic modifications. Decreased liver mtDNA content concomitantly contributes to peripheral IR.

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Section: Methodsmentioning

confidence: 99%

Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: Impact of liver methylation of the peroxisome proliferator-activated receptor γ coactivator 1α promoter

et al. 2010

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“…The primer pairs for amplifying unmethylated DNA for the region Ϫ137 to ϩ53 were 5Ј-TTTATTTGGTTTGTTTGTTGTTGTT-3Ј (forward) and 5Ј-ACTCCATACTCTCTACCCATCATAA-3Ј (reverse). These primers, which encompass the CpG island of the DR3 promoter, were searched with MethPrimer software (available at http://www.urogene.org/methprimer/ index1.html) (25).…”

Section: Methodsmentioning

confidence: 99%

Hypermethylated promoter region of DR3, the death receptor 3 gene, in rheumatoid arthritis synovial cells

Takami

Osawa

Miura

et al. 2006

Arthritis & Rheumatism

139

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Objective. To examine the promoter activity and protein expression of the death receptor 3 gene DR3, a member of the apoptosis-inducing Fas gene family, with particular reference to the methylation status of its promoter region in rheumatoid arthritis (RA).Methods. Genomic DNA was prepared from peripheral blood mononuclear cells obtained from healthy individuals and from patients with RA and synovial cells obtained from patients with RA and osteoarthritis. The methylation status of the DR3 promoter was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction techniques. Gene promoter activity and protein expression were examined using the luciferase reporter and Western blotting techniques.Results. The promoter region of the DR3 gene contained many CpG motifs, including one CpG island that was specifically hypermethylated in synovial cells from patients with RA. Promoter assays showed that the promoter CpG island was essential for the transactivation of the DR3 gene and that forced hypermethylation of the CpG island with the bacterial methylase Sss I in vitro resulted in inhibition of the DR3 gene expression.

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“…PCR was performed using Platinum Taq DNA Polymerase (Invitrogen) along with either the specific methylated (M) or unmethylated (U) primers. Primers were generated using MethPrimer [29] with the parameters set for optimal results as follows: product size 200 bp, Tm 55°C and primer length 25 bp. For methylated BRMS1 specific PCR, the primers utilized were: forward-5′ TAG ATG TTT TAC GTT ATT CGG TGC 3′ (−2461 to −2436) and reverse-5′ ATT AAT CTT ACT CCT CCT ACC CGT A (−2356 to −2337).…”

Section: Methylation Specific Pcr (Msp)mentioning

confidence: 99%

Epigenetic silencing contributes to the loss of BRMS1 expression in breast cancer

Metge

Frost

King

et al. 2008

Clin Exp Metastasis

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Breast Cancer Metastasis Suppressor 1 (BRMS1) suppresses metastasis of human breast cancer, ovarian cancer and melanoma in athymic mice. Studies have also shown that BRMS1 is significantly downregulated in some breast tumors, especially in metastatic disease. However, the mechanisms which regulate BRMS1 expression are currently unknown. Upon examination of the BRMS1 promoter region by methylation specific PCR (MSP) analysis, we discovered a CpG island (−3477 to −2214), which was found to be hypermethylated across breast cancer cell lines. A panel of 20 patient samples analyzed showed that 45% of the primary tumors and 60% of the matched lymph node metastases, displayed hypermethylation of BRMS1 promoter. Furthermore, we found a direct correlation between the methylation status of the BRMS1 promoter in the DNA isolated from tissues, with the loss of BRMS1 expression assessed by immunohistochemistry. There are several studies investigating the mechanism by which BRMS1 suppresses metastasis; however thus far there is no

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MethPrimer: designing primers for methylation PCRs

Cited by 2,309 publications

References 20 publications

Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: Impact of liver methylation of the peroxisome proliferator-activated receptor γ coactivator 1α promoter

Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: Impact of liver methylation of the peroxisome proliferator-activated receptor γ coactivator 1α promoter

Hypermethylated promoter region of DR3, the death receptor 3 gene, in rheumatoid arthritis synovial cells

Epigenetic silencing contributes to the loss of BRMS1 expression in breast cancer

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