title: Disclosing bias in bisulfite assay: MethPrimers underestimate high DNA methylation.
abstract: Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated) versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of "Methylation-Insensitive Primers" (MIPs), having degenerated bases (G/A) to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies.
authors.affiliation: Dept. of Psychology, Sapienza University of Rome, Rome, Italy; European Center for Brain Research (CERC)/IRCCS Santa Lucia Foundation, Rome, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Fuso
authors.affiliation: Dept. of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy.
authors.forename: Giampiero
authors.initials: G
authors.lastname: Ferraguti
authors.affiliation: Dept. of Surgery "P. Valdoni", Sapienza University of Rome, Rome, Italy.
authors.forename: Sigfrido
authors.initials: S
authors.lastname: Scarpa
authors.affiliation: Institute of Neuropathology, IDIBELL-Bellvitge University Hospital and University of Barcelona, CIBERNED, L'Hospitalet de Llobregat, Spain.
authors.forename: Isidre
authors.initials: I
authors.lastname: Ferrer
authors.affiliation: Dept. of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy; Pasteur Institute, Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Lucarelli
mesh_terms: D000544:Alzheimer Disease / Q000235:genetics / Q000473:pathology*; D000818:Animals; D002460:Cell Line; D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers / Q000032:analysis*; D006801:Humans; D051379:Mice; D018008:Myogenin / Q000235:genetics*; D053764:Presenilin-1 / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software; D013447:Sulfites
chemical_list: D017931:DNA Primers; D018008:Myogenin; D053764:Presenilin-1; D013447:Sulfites; C042345:hydrogen sulfite