Video: Journey of a cancer sample, part 4
Every drop of DNA derived from a patient’s tumor is precious.
Mark Puppo and Kristin Anderka are highly aware of how much time and effort has been devoted to each of the samples they are examining today. As you will see in the video below, they take great care in handling each sample and all of the chemicals in their section of the laboratory.
For several weeks, we have been following lung cancer samples on their journey through the Broad’s Biological Samples Platform (check out previous steps of this journey, here, here, and here). Last week, DNA was isolated from these samples, and now it is time for two critical steps: “quantification” and “normalization.”
Quantification is the process of determining how much DNA is in a given sample, and normalization is the process by which Mark and Kristin dilute the samples to get them into a proper concentration range and to ensure that there will be enough material for later steps.
“A massive amount of work goes into each sample before it arrives in my hand,” says Mark. “What is actually in each test tube ends up being about a tenth of a milliliter – a very small volume.”
Each laboratory action that Mark and his colleagues perform uses up between two and four percent of the sample. “You can deplete a sample quite quickly if your processes don’t go as well as you had hoped,” says Mark. “We try to get it right the first time, which is where dilutions come in.”
Diluting the sample makes certain that the source material will not be exhausted over the course of the sample’s journey. Mark does some calculations to determine a dilution factor for the samples. These dilutions can be done by hand, but using a robot known as a liquid handler can save time and also decreases the chance of human error.
Mark then transfers two microliters of the diluted material onto a “microfluor” plate. He adds a fluorescent dye called PicoGreen that binds to double stranded DNA and runs the samples through a machine called a spectrometer, which measures how strongly each sample glows to determine DNA concentration. He records all of this information in an electronic database so that the data will travel along with the sample wherever it goes.
At this stage, the cancer samples could be stored in BSP’s vast freezers, where thousands of samples lie in wait. But these lung cancer samples have been fast-tracked – their journey is about to pick up speed. When we return next time to BSP, it will be the last time for a while. The samples will be carefully prepared by one last BSP researcher and then handed off to another Broad team for the beginning of genetic analysis.