Dr. Liu from China stays in TMDU and NCCRI, Japan: Part2
A3 Foresight Program: Education Program for Young Researchers
Report of visit to Department of Molecular Oncology in TMDU and Division of Epigenomics in NCCRI (Nov. 28-Dec. 9, 2011)
Zhaojun Liu (Department of Cancer Etiology, Peking University School of Oncology)
Host researcher
Prof. Yasuhito Yuasa, M.D Ph.D (Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University) and Dr. Toshikazu Ushijima, Ph.D. (Division of Epigenomics, National Cancer Center Research Institute)
Summary
From Nov. 28 to Dec. 9, 2011, I had a study stay at Department of Molecular Oncology in TMDU and Division of Epigenomics in NCCRI. The main purposes of this stay were to practice the protocol to culture a gastric cancer cell line, and to carry out independent validation study on the relationship between the methylation status of three genes and metastasis of gastric carcinomas. I deeply appreciated this chance to visit Prof. Yuasa’s and Dr. Ushijima’s laboratories for the cooperative study. At the same time, I got great experiences and wonderful memories from Japanese friends.
Contents
1. Culturing a gastric cancer cell line
In our previous work, we found that gene A was partially methylated in a gastric cancer cell line. I plan to use this cell line to analyze biological and functional changes resulted from gene methylation in my further study. The detailed protocol to culture this cell line is listed below.
A. Cell thawing
☆ Remove vials from liquid Nitrogen;
☆ Add 3mL medium to a 10mL conical tube;
☆ Transfer contents of cryogenic vial to the conical tube and spin at 1000rpm for 5 min;
☆ Discard medium and re-suspend pellet in 4mL fresh medium;
☆ Transfer cells to a collagen-coated flask and incubate at 37ºC with 5%CO2.
B. Cell passage
☆ Digest the cells by Trypsin (0.025%) for 1-2 minutes at room temperature;
☆ Carefully transfer the digested cells into the centrifuge tubes and centrifuge at 1000rpm for 5 minutes;
☆ Remove the supernatant;
☆ Add 1ml fresh medium and gently mix the cells;
☆ Carefully transfer the cells back to the flask or plate.
2. Sodium bisulfite treatment
☆ DNA denaturation;
☆ Sodium bisulfite treatment via cycling reaction system containing NaHSO3, NaOH and hydroquinone;
☆ DNA purification by Zymo-spin column.
3. Confirmation study on 3 genes’ methylation and metastasis of gastric carcinoma
According to our previous work, the methylation status of the three genes was correlated with metastasis of gastric cancer. It would be more valuable if this result could be confirmed in more samples. So we carried out the validation study using more gastric cancer samples from 130 patients at Prof. Ushijima’s lab. at Division of Epigenomics in NCCRI.
4. Quantitative methylation-specific PCR of gene A with standard and sample DNA
Standard DNA was diluted into different concentration as the control to calculate the copy number of the samples.
5. Presentation of my work in NCCRI
I gave a presentation of my work titled “Methylation of gene A predicts less metastasis of gastric carcinoma” in Division of Epigenomics, NCCRI on Dec. 2. Dr. Ushijima and other laboratory members attended the meeting and gave me some good suggestions about my work.
Acknowledgments and a photo
I sincerely thank Prof. Yuasa and Dr. Ushijima for kindly reception. I thank Dr. Akiyama and Dr. Hattori not only for the thoughtful arrangement and patient instruction of the experiment but also for the warm concern of my daily life. I thank Dr. Fukamachi and Dr. Shimada, Dr. Shigematsu and other lab members in both laboratories for the generous helps and warm hospitality.
