Maldi tof mass spectrometer

Haematoxylin and eosin stained sections of each case were reviewed by two pathologists; tumours were staged and subtyped according to WHO Classification of Tumours of the Lung [32 ]. DNA was extracted from representative 5-µm-thick sections cut from formalin-fixed and paraffin-embedded blocks of each tumour sample to ensure tumour cell content is above 20% as previously described [33 (link)]. Genomic DNAs were subjected to a mutation survey using the LungCarta panel (Sequenom), including evaluation of 214 somatic mutations in 26 oncogenes and tumour suppressor genes, and analysed on a matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometer (Sequenom) following manufacturer’s protocol. Data were evaluated using MassARRAY TYPER ANALYSER software 4.0, with a limit of detection of 5%.

DNA methylation level of the IRF8 promoter was quantified by using Sequenom MassARRAY EpiTYPER analysis using the following procedure: the initial denaturation at 95 °C for 4 minutes, denaturation at 95 °C lasts 20 seconds for 45 cycles, then annealing at 58 °C for 30 seconds, extension at 72 °C for 1 minute, and then incubation at 72 °C for 3 minutes. The primers employed for IRF8 methylation analysis were designed for 195 bp of the MassARRAY® EpiTYPER (Sequenom, Inc., CA, USA). The analyzed region of IRF8 is located −441 bp to −225 bp up-stream from the transcription initiation site (TSS) of the first exon. Methylation primer of IRF8 was as shown: forward: 5′-aggaagagagGGGTAGTTAGTTTTTGGTTGTGGAT-3′; reverse: 5′-cagtaatacgactcactatagggagaaggctTACAAAAAAACTTTCCCAAAAATTC-3′. The shrimp alkaline phosphatase (SAP) and MassCLEAVE reaction were then performed. The end-products were desalted and dispensed to a SpectroCHIP, using a MassARRAY™ Nanodispenser (Sequenom, Inc., CA, USA). A MassARRAY compact matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Sequenom, Inc., CA, USA) was used to acquire the spectra. The methylation results were analyzed with EpiTyper software (Sequenom, Inc., CA, USA).