Sequenom genotyping platform

For this part of the study three different techniques were used. Genotyping of the G594A variant in the ESR1 gene was carried out by using PCR-RFLP. The restriction enzyme BtgI (New England Biolabs, Australia) was used for the determination of the SNP genotype. Variant C325G (rs1801132) was genotyped using TaqMan^® SNP Genotyping Assay (life technologies, Cat. #4351379) and following the manufacturer instructions.
PROGINS insertion/deletion was tested with a standard PCR. Samples were then observed on a 2% agarose gel to detect the presence of the PROGINS insert (details in Additional file 1).
A total of 34 SNPs were selected from 14 different genes on nine chromosomes as the research panel for the study. Selected SNPs were located in genes involved in neuronal, hormonal and immunologic pathways previously associated with migraine. The SNPs were tested by using the Sequenom genotyping platform (Sequenom^®, San Diego, CA, USA), which uses MALDI-TOF mass spectroscopy and MassARRAY technology with an iPlex system. Primers for polymerase chain reaction (PCR) amplification and single base extension were designed by Sequenom Assay Design 3.1 software (Sequenom, San Diego, CA, USA) according to the manufacturer’s instructions (Additional file 2).