Esophageal Adenocarcinoma Methods and Protocols

237 Mix thoroughly by pipetting up and down five times, then transfer master mixes to two wells of a 96-well PCR plate (Table 1 ...

238 As multiple samples libraries will be loaded for each sequenc- ing chip, each sample must be assigned a unique barcode to e ...

239 Carefully remove the plate seal, and then add the following components in the order listed to each well containing digested ...

240 75 μL of freshly prepared 70% ethanol should be added (com- bine 230 μL of ethanol with 100 μL of nuclease-free water per s ...

241 Dilute library to ~100 pM, combine, then proceed to template preparation, or libraries can be stored at 4–8 °C for up to 1 ...

242 The Ion Sphere Particles (ISPs), included in the template preparation kit, should then be vortexed at maximum speed for 1 m ...

243 The Personal Genome Machine (PGM) (Fig. 4a) should be maintained including weekly chloride washes when the machine is in us ...

244 The run plan specified sequencing settings, number of flows, kit type used in sample preparation, barcodes used and corre- ...

245 by the quality filters for being polyclonal, having low read quality, or the result of primer dimer. These reports are used ...

246 drying the beads. Under conditions of low relative humidity, the beads air-dry rapidly. Do not overdry. The enriched ion sp ...

247 Alfred K. Lam (ed.), Esophageal Adenocarcinoma: Methods and Protocols, Methods in Molecular Biology, vol. 1756, https://doi. ...

248 DNA hypermethylated mediated loss or reduced expression of genes involving in cell cycle regulation, DNA and growth factors ...

249 for DNA methylation is 5′-CpG-3′ dinucleotides (CpG islands). CpG islands are small stretches of DNA about 300–3000 bp, whic ...

250 Bisulfite conversion is the most commonly used method for study- ing DNA methylation in which the non-methylated cytosines a ...

251 of interest can identify the methylation status compared with the controls. The result of PCR amplification of the bisulfite ...

252 Gel staining reagent. Gel loading dye. Plasmid construct for cloning the PCR products. Universal methylated and unmethylate ...

253 (d) Incubate the mixture using a thermal cycler with the fol- lowing condition: two cycles with 95 °C for 4 min, and then 55 ...

254 of bisulfite-converted DNA, 2 μl of RNase free water, and 1 μl of forward and reverse primers (see Note 5). Run the PCR wit ...

255 References Suzuki MM, Bird A (2008) DNA methylation landscapes: provocative insights from epig- enomics. Nat Rev Genet 9:46 ...

256 Methylation of SOCS-3 and SOCS-1 in the carcinogenesis of Barrett’s adenocarcinoma. Gut 56:1047–1053 Jin Z, Olaru A, Yang J ...