The study showed LOI of IGF2 is associated with gastric corpus and LNM in gastric cancer tissues, suggesting that IGF2 plays an important role in gastric carcinogenesis. Methods Tissues and information collection The panel of gastric tissues consisted of paired fresh normal adjacent-tumorous and tumorous specimens from 89 learn more GC patients during surgery before any other treatment in the Department of oncology, China Medical University affiliated the first Hospital from March 2007 to February 2008. Written informed consents were obtained from all patients. Demographic and clinicopathologic

information were collected from each patient. Tumour location was classified as gastric antrum, gastric corpus, gastric cardia cancer. The tissues were frozen immediately

in an -80°C freezer until use. Nucleic acid preparation After homogenizing the frozen tissues, genomic DNA was extracted using standard procedures with phenol/chloroform and precipitated with ethanol. RNA was extracted from grounded tissues using guanidinium isothiocyanate-phenol solution (RNAzol B, Biotecx Laboratories. Inc., Houston, TX, USA) following the manufacturer’s instructions. RNA was treated with Oligomycin A concentration Rnase-free DNaseI to eliminate DNA contamination (BRL, Baltimore, MD, USA) and stored at -80°C until use. Analysis of informative LIT1, IGF2 and H19 cases Firstly, analyses were performed from DNA of normal tissues to determine informative cases. Heterozygosity in the LIT1, IGF2 and H19 gene was determined by the presence or absence of RsaI, ApaI and HinfI, and RsaI sites this website respectively. Informative genomic heterozygotes for the LIT1, IGF2 and H19 were studied as follows. The polymorphic region of LIT1, IGF2

and H19 were amplified with the primers [25, 18] The PCR reaction was conducted in 1 × PCR buffer with 1 μm primers, 200 μm dNTP, 2.5 units Taq DNA polymerase (Perkin-Elmer, Foster City, CA, USA) and 200 ng genomic DNA. Conditions for amplification were 94°C for 2 min followed by 30 cycles at 94°C for 30 sec, 54 c, 56°C and 58°C (for the LIT1, IGF2 and H19 respectively) for 1 min, and 72°C for 1 min. A final step was 72°C for 5 min. The PCR products were subject to RsaI, ApaI and HinfI and RsaI (New England Biolabs, Beverly, Mass, USA) enzyme digestion at 37°C overnight, run through 12% acrylamide gel Idelalisib and stained with ethidium bromide respectively. The expected size of the PCR fragment of the LIT1 gene is 410 bp. Informative heterozygous cases exhibit three bands of 188, 222 and 410 bp. For IGF2 Primers P1 and P3 were also used to get a 1.4 kb DNA fragment that was used as a size control for the RT-PCR product. PCR conditions were the same except for a 1.5-min annealing step at 60°C C with primers P1 and P3. The PCR products for IGF2 resulted in a 292 bp band with primers P2 and P3. Informative cases are those in which one allele had an ApaI restriction site (256 bp) and the other had an HinfI restriction site (231 bp).