Week 5 – Fecal Samples and Iodine

As I mentioned in my past post about my project, I am determining the thyroid concentration in fecal samples of a wild population of male baboons using T3 radioimmunoassay (RIA).

First, here’s a bit more information about how RIAs work: In a T3 RIA, an unknown amount of T3 thyroid hormone competes with a known amount of radioactively labeled T3 for binding sites on an antibody-coated tube. The antibody has equal affinity to the radioactive T3 tracer solution (in which the T3 is labeled with 125I) and the T3 present in the fecal samples. Once the T3 in the fecal sample competes with the T3 tracer solution for the binding sites, the antibody-coated tubes can be placed in a gamma counter that will determine radioactivity of the sample, which is measured in counts per minute (cpm). Seven standards (A-F and B ½) that contain different, known concentrations of T3 are analyzed each time a RIA is performed so that a standard curve can be generated. Therefore, the radioactivity of the fecal samples can be compared to the standards, and the concentration of T3 can be extrapolated from the curve.

After five weeks of working on my research project, I have performed 22 RIAs on about 1,500 fecal samples. Since the results of my project heavily rely on how many samples my mentor and I analyze, so far my days in the lab have followed a fairly predictable routine.

When I arrive in the morning I first collect the pre-selected 68 samples sitting in the freezer and prepare them for analysis the following day. To prepare them, I warm the samples to about 37°C in a water bath, vortex them for 10 seconds (more often than not there are solid components at the bottom of the test tube and we need those to be evenly distributed throughout the solution), transfer 2.5 mL of each sample into test tubes, and then dry them in the evap-o-rack. All the steps leading up to the evap-o-rack can take about 1.5 to 2 hours to complete. While the amount of time it takes for the samples to dry can vary, once the samples start drying then the morning phase of my day is over and onward I progress to the next stage of my day: performing the RIAs. (An important note: once the samples are completely drying sometime during the afternoon, I store them in the freezer to be analyzed the following day.)

In the second, longer part of my day, I retrieve the dry samples prepared the prior day and add 250 μl of Standard A (which contains no T3) to every test tube. I then vortex all the samples for 30 seconds, transfer them into Eppendorf tubes, and obtain the antibody-coated tubes. 100 μl of each standard, three controls, one pool, and the samples are added to two tubes as outlined below, ultimately resulting in 160 tubes that will be put in the gamma counter. But before I reach that step, I add 1 ml of the T3 tracer to all the tubes, vortex them for 10 seconds, incubate the samples for 1 hour at 37°C, aspirate the tubes, add 3 ml of deionized water to them all, then aspirate again. After the tubes are aspirated for the second time, I place them into the gamma counter and voilà: my day in the lab is over.

Before I leave, I like to label the test tubes and Eppendorf tubes to prepare for the next time I’m in the lab, which helps me save some time during the process. In the first couple of weeks, I often would end up leaving lab after 7:00. By week 4, I got use to the procedure and became more efficient, which helped me settle in a regular schedule of work, have a snack, work, have lunch during the hour wait, then finish up for the day and leave around 6:00. The only exception to this flow is on Mondays when I join my lab for the weekly lunch meetings. I may end up leaving the lab after 7:00 on Mondays, but it’s well worth it to spend the time with members of the Alberts lab and my mentor’s two dogs: Cyclone and Twister.

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