Methylation Analysis of HTR2A Promoter

Two CpGs (−1420 and −1224) were studied based on findings of Falkenberg and colleagues (2011) (link) and Paquette and colleagues (2013) (link). Saliva samples were obtained using the Oragene DISCOVER kits (OGR-575) for Assisted Collections (DNA Genotek, Kanata, Ontario, Canada), and DNA was isolated following the manufacturer’s instructions. A 276 bp. region of the HTR2A promoter region (Chr 7: 28415063-28414801) was amplified from bisulfite modified DNA using the pyromark PCR kit (Qiagen, 203443), and DNA methylation assessed through pyrosequencing following a serial pyrosequencing protocol using a PyroMark MD system (Qiagen) and two sequencing primers as described in Paquette et al (2013) (link). Each pyrosequencing assay contained a bisulfite conversion control to assess conversion efficiency. All samples examined demonstrated >95% conversion.

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Parade S.H., Novick A.M., Parent J., Seifer R., Klaver S.J., Marsit C.J., Gobin A.P., Yang B.Z, & Tyrka A.R. (2017). Stress exposure and psychopathology alter methylation of the serotonin receptor 2A (HTR2A) gene in preschoolers. Development and psychopathology, 29(5), 1619-1626.

Publication 2017

Oragene DISCOVER kits pyromark PCR kit PyroMark MD system

Assay Bisulfite Cpgs Dna methylation Htr2a Primers Saliva

Corresponding Organization : Bradley Hospital

Other organizations : Butler Hospital, Florida International University, Emory University, Yale University

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Variable analysis

independent variables

Two CpGs (−1420 and −1224)

dependent variables

DNA methylation

control variables

Bisulfite conversion efficiency (all samples examined demonstrated >95% conversion)

controls

Positive control: Bisulfite conversion control to assess conversion efficiency
Negative control: None explicitly mentioned

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