Friday, August 20, 2010

Week 7 + a few days in August

The last week of summer immersion (plus a few days in August), provided an opportunity to see the robotic lab where all of the patient samples are tested in the hospital, time to collect images from my nearly 50 histology slides, a lesson in selecting primers, and a chance to run a qPCR on the lung tissue I'd harvested from three sets of mouse lungs.

A visit to Robo Lab was a great way to start the week. As someone who is interested in high-throughput operations, this lab offered a glimpse into the way hundreds of thousands of samples can be tracked and processed every day. Although the system works fairly well, there are still major issues that need to be addressed, and the efficiency should go up significantly with renovations and upgrades scheduled for next year. Some of the problems with the current system were obvious (e.g. physically moving samples across long distances rather than drawing multiple samples from tubes in a single location), while other problems were more difficult to spot (e.g. using mechanical tracks that are prone to malfunctioning or contain parts that readily wear out, and using systems from multiple suppliers/manufacturers so cohesiveness becomes an issue and technical support can be difficult to receive). It was also interesting to see the way the samples are transported through the hospital via a vacuum tube system. It would be nice to see the updated and condensed Robo Lab a few years from now.

Before selecting the genes of interest for my qPCR, I spoke with a researcher in the lab who has done studies on wound healing in mouse lung and liver samples. Based on our conversation, nearly a dozen genes were identified for analysis. The first step in ordering primers is to consult the National Center for Biotechnology Information (NCBI) database in order to identify the loci of genes as well as to find the sequence. The Harvard Primer Bank is then used to identify primers that others have reported as being useful when amplifying the gene of interest. Since others in the lab will likely use these stocks of primers for cell cultures containing both human and mouse cells, it is important to use the Basic Local Alignment Search Tool (BLAST) to verify that both the forward and reverse primer for each gene is a 100% match for the mouse gene and at least one of the two primers is not a match for the human gene. Once this is done, the primer can be ordered. Once the primers arrived, the stocks were diluted to a standard concentration, the concentration of the RNA was analyzed, cDNA was made from the RNA, and the qPCR was run. Eleven genes were analyzed along with one house-keeping gene for normalization of the results.

A preliminary review of the data has revealed which genes were up regulated and which were down regulated as a result of 45 hours in the bioreactor, and a third tissue set, involving a different media, has provided even more interesting results. Unfortunately, the company hold the components of the media as a trade secret, so it will be difficult to form a strong hypotheses as to why the change in media resulted in such a dramatic change in genetic regulation.

Summer Immersion has been a wonderful experience that has helped shape my outlook on biomedical engineering. Thank you to everyone who has helped make this possible. I look forward to hearing about the experiences next year's class will have during immersion.

No comments:

Post a Comment