The National Science Foundation's (NSF) Tokyo Office periodically receives and disseminates reports on research developments in Japan that are related to the Foundation's mission. NSF-sponsored researchers currently working in Japan prepare many of these reports. These reports present information for use by NSF program managers and policy makers; they are not statements of NSF policy.
Special Scientific Report #00-12 (November 16, 2000)
Mr. Paul J. Nordone, Jr., a graduate student in Natural Sciences at the Roswell Park Cancer Institute at the State University of New York at Buffalo, prepared the following report. Mr. Nordone was a participant in the 2000 Summer Institute sponsored in the United States by NSF/NIH/USDA and the Science and Technology Agency and Japan Science and Technology Corporation in Japan. Dr. Yasushi Okazaki at the RIKEN (Institute of Physical and Chemical Research) Biosciences Center in Tsukuba, hosted Mr. Nordone. Mr. Nordone can be reached via email at: nordone@usa.net.
This program was packed with so many wonderful experiences–both cultural and educational. The language training was sufficient for me to travel outside of the major cities into the vast wilderness where speaking the language was an absolute must, or primitive cave-man drawings would be your only means of communication!
The technology that I learned during my two months of research at RIKEN had no connection to my current Master’s degree; however, this DNA chip (microarray) technology is a field that I feel will revolutionize today’s world of medicine and scientific research. I’m very excited to have learned about this technology and am looking forward to pursuing a career within a related field of biotechnology following completion of my degree.
What is a microarray?
An array is an orderly arrangement of samples. It provides a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identifying the unknowns. I worked with DNA microarrays, or DNA chips fabricated by high-speed robotic machines. These robots generally use glass slides for deposition of the DNA, although nylon substrates are also used. Probes with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies. An experiment with a single DNA chip can provide researchers information on thousands of genes simultaneously - a dramatic increase in throughput, compared to the Northern hybridizations that I performed during my Master’s research, which give information on only one gene at a time. This technology will facilitate the pharmacogenomics industry, where one can develop drugs based on the individual differences in a person’s genetics. Microarray technology will eliminate unnecessary deaths and hospitalizations from adverse prescription drug interactions while visualizing the effects that a single drug can have in altering the expression of numerous genes.
At the Lab Bench
I sacrificed some NOD mice (a non-obese diabetic inbred strain). My project was to examine the differences in gene expression on the liver tissues of different sex and age groups (4wk and 8wk). My control was an equal mixture of male and female 17.5 day embryos of a B6 strain. I got great yield from my RNA preps using a slightly different protocol than my past–only one phenol/chloroform extraction, and CTAB to remove polysaccharides. After ethanol precipitation to concentrate the RNA, I labeled the samples with Cy3 and Cy5-UTP fluorescence by reverse transcription with Superscript II, and hybridized the samples to the microarrays. Only 9ul of hybe solution are necessary for each multiblock containing 7,000 genes–(good thing since each experiment on each age group runs about $1,000!) The 9ul is placed on the edge of a cover slip and carefully positioned over the slide containing the multiblock of genes. The full-length mouse cDNAs are printed on a glass slide–two cover slips/slide–1 1/2 slides for a total of 21,000 genes. Currently, work is proceeding on a new set of cDNAs and will expand to 42,000 genes/3 slides. After a 12-hour hybridization, the slides are washed, dried, and ready for analysis. The analysis of the slides was done on ScanArray 5000 by GSI Lumonics, and Scanalyze Software, although I've seen a software program in the lab by MolecularWare (costs $10,000) that automatically moves the grid to fit each individual spot perfectly. I’m looking forward to processing and publishing the results after completion of my Master’s thesis.
Problems with the Microarrays
Since this technology is so new, there are numerous problems with its use. First is the difficulty in distinguishing the differences is genes expressed at a very low level. There are clearly numerous genes with expression differences of over 40 times the control, but many hormones and proteins in our body function at extremely specific and low levels. Another problem with the microarray procedure deals with the fluorescence dye, Cy3 and Cy5. There is a bias in the incorporation of these dyes due to differences in their chemical structures. This may give a false positive (or negative) in fluorescence signal. One way to resolve this bias is the fluoro-reverse method. I labeled my controls with Cy3, and samples with Cy5, in duplicate, and also reversed the labeling of the control with Cy5, and samples with Cy3, yielding four experiments for each sample. This greatly reduces the bias of dye incorporation, although a new method of using the amino-allele compound causes the dyes to incorporate at a much more equal and greater amount, reducing bias and increasing sensitivity.
After the Program
One of the main goals of this program is to facilitate communication between Japanese scientists and foreign researchers. Just one week after our program ended I was doing just that. I was invited to participate in the FANTOM meeting (Functional ANnoTation Of Mouse), held at RIKEN, the institute where I just completed my summer research. Fifty well known scientists from all over the world flew in to help in naming 20,000 of RIKEN’s full-length CDNA’s. Many of these scientists were from very well-respected laboratories including The Jackson Lab, Genome Institute of Novartis, Laboratory of Genetics NIMH/NHGRI, EBI (European Bioinformatics), Rockefeller University, Lion Bioscience, TIGR (The Institute of Genomic Research), NCBI and Harvard. In order to accomplish this feat within a two-week time frame, several of Riken’s top bioinformatists developed an annotation tool, combining many of the best DNA and protein search engines like Interpro, Blastn, Blastx, Swiss Prot, Bioscout, and Orf finder on to one web page. This made the annotation process much easier and smoother; however, the rules of the annotation and use of the interface still had to be explained to the scientists. On August 28, 2000, the first day of the meeting, I was chosen to help explain to the participants how to use this web interface. I was very fortunate to have participated in this venture with these well-known and respected scientists in the field of genomics.
Traveling through Japan
After the FANTOM meeting, I activated my JR (Japan Rail) Pass (a must if you want to travel and save money). I visited the cities of Kyoto, Nara (where deer, right on the streets would eat out of your hand and lick the side of your face like a dog), Hiroshima, and Fukoka, the last stop on the Shinkansen (bullet train). Fukoka is in Kyushu, the southernmost of Japan's four principal islands, where I went to the Takeo Onsen and the live volcano, Mt. Aso. From Fukoka, I took the bullet train all the way to Japan’s untouched wilderness in Hokkaido, the northernmost of its major islands. I briefly stopped in Hakodate, a beautiful city with the freshest seafood, but skipped over Saporro, the capital of Hokkaido, and headed east towards Shari. I reserved the overnight train so I wouldn’t lose any time traveling during the day. My first stop was this hot springs waterfall that I heard about from a friend in my lab, Kamuiwakka Takki. It was the most amazing site I’d ever seen, as hot water from the earth’s interior splashed down into a pool of water deeper than you could touch the bottom. Next I hiked up Mt. Laos into the clouds, and spent the night at the bottom in Iwabetsu Onsen. Hitchhiking is easier than waiting for the bus in this area, as there are only two per day, and I was lucky enough to find someone going to the same place as me, Lake Moshu. This is the cleanest most pristine lake in all of Japan, and the fog lifted so you could see the lake’s island. After that, it was my trip coming to an end, and my last meal in Japan was a king crab that I hand picked and brought to the restaurant to cook. Yum!
All in all, my trip was amazing. The only down side was an auto accident I was in during the time I was working in the lab at RIKEN. Obviously, the accident didn’t stop me from finishing off my trip with traveling and hiking, but needless to say, I am very fortunate–fortunate to not only be alive and well, but fortunate to have had the opportunity to participate in this most wonderful program.