The Telomeres

Hello and welcome to the final blog!

This week, we resolved our issues, made great headway, and ate some lunch while we were at it.

Firstly, we definitely corrected the issue from last week of high density v. low density. I plated a lower density experiment to be analyzed, and the results have been interesting (but that’s for the presentation). Luckily, we were able to use the high density results for the good of the project, instead of letting it go to waste. The higher density, although it did not show quite as much of a reduction in the MYC protein which actually drives the cancer and causes proliferation, showed a definite reduction in the IRF4 protein, which is the protein the drug was meant to target. If you remember, in previous blogs I mentioned that IRF4 drove the production of MYC in multiple myeloma. So, the purpose of the drug was to target IRF4, reducing it so that MYC would subsequently be reduced and the cancer would die off. So, what we saw is that the drug was working—but that, in high density settings, there still remained enough IRF4 for the MYC to be produced and the cancer to continue its proliferation.

So, when we began experimenting on the lower density cells, we saw a dramatic decrease in cell proliferation after 6 days of drug treatment (a promising sign that MYC was knocked down). However we also got some strange results after putting the cells through some flow cytometry, which analyzes each cell in a sample individually. And those results should be revealed in the presentation.

Until then, thank you for reading!

Angela

It's a Friday

Hello and welcome back!

This week at the Mayo Clinic has been quite eye-opening, especially when one realizes that one has been conducting an experiment wrong for the last two weeks.

Nevertheless, at the start of the week, everything was going smoothly. We were repeating the western blot, which had a strange result (it showed no effect on the cell lines) and we were midway through the RNA Analysis.

By the second day, the western blot was ready to be put into a gel to be read and we were using the PCR machine to make more RNA for later testing.

And then, come Friday, we had finally finished the Western Blot and had already put the RNA into the gel, when we decided to look back at the protocol and I realized that everything was wrong.

Well, not everything. I exaggerate. But, it happens to be a slight problem when you plate the cells at 5 times the concentration they’re meant to be at. If you recall, one of the first problems the experiment had run into when we were first analyzing it was that the drug did not work under high concentrations of cells. The cells are meant to be at a low concentration of 50K cells/ml and I, in all my infinite wisdom, had been plating the most recent cells at 250K cells/ml.

Oops.

Luckily, it’s easily solved and doing the experiments a third time over just means I’ll have a more experienced hand, right?

Right.

Thank you for reading!

Angela

Lab, lab, LAB!

Hello and welcome back to the blog!

This week, I did much more of the same: RNA and protein analysis, excel graphs, and presentation slides. Our results have been very consistent so far, and we’re seeing a confirmation of what we expect to be happening theoretically in every step of the DNA to protein synthesis. The graphs, by far, are still the most time consuming part—both in the way of examining the data and plotting it. Did you know about the log scale function? I do now…

But, as a small change of events, I was able to go to one of the lab meetings this week. Usually, the lab meetings are on Fridays and are a short presentation of our work between the 5 of us in the lab. So, I should have known something was odd when I found out there was a lab meeting Monday, instead. But, at the time, I didn’t. And when I arrived at the lab meeting and there were buckets of food outside the door, I should have thought that was odd as well. But, I didn’t and I happily picked up a veggie sandwich. It was really only when I opened the door to the meeting room and 6 times the amount of people I was used to seeing that I knew something was up.

Evidently, this would be the mother ship of meetings: a discussion by all the scientists working on Multiple Myeloma in the Mayo Clinic Labs. A lot of the information that was transferred between this groups passed over my head: but what little I did gather was incredible and encouraging. There are many new connections to be made about multiple myeloma that will allow us to understand it more. It was more than exciting to see so many so passionate about this one field, and to witness the joining of like, experienced minds.

Thank you for reading!

Angela

Don't Cell Out

Hello, and welcome back!

This week, we found some of our results and everything in the way of the lab seems to be going smoothly and consistently.

Things in the way of presentation, however, are not going so smoothly.

To start off, the drug titration showed the same results as the test on the drug proliferation. It seems that the cells tend to drop off at a drug concentration of around 1uM.

The western blot also showed consistent data. We saw that the MYC protein was reduced once the active drugs were added, compared to the inactive drugs. Unfortunately, we will have to repeat the experiment since the staining did not turn out quite as clearly as we would have liked.

And finally, the analysis of the RNA is in progress, and coming along smoothly.

Powerpoint, however, has not been so easy. And Excel, its analytical cousin, has been equally ornery. I’ve been attempting to create diagrams and graphs that would present the data with the elegance it deserves, and, unfortunately for everyone, the best I could do was this:

Obviously, this isn’t going to work. So, I’ve been trying to enlist the help of this strange thing called “google” to get me to a better diagram. Unfortunately, every time I google “cells”, I’m either taken back to fifth grade science or a man in black and white stripes. On the bright side, I have a few more weeks to become a professional in the art of Office.

Until then, thank you for reading!

Angela

Juggling

Hello and welcome back!

This week I learned to juggle… that is, I learned to juggle multiple new experiments and strange new jobs.

To start off, though it may seem a little unrelated, I’ve begun working at McDonald’s. It’s been interesting to say the least. Luckily, it hasn’t affected my schedule at Mayo Clinic, although it has altered my outlook a little bit. I think I can appreciate the slow, relaxed, meticulous pace of the lab more now that I can compare it to swearing customers, burning French fries, and toppling ice cream cones. However, as different as it may seem, I’ve been able to draw some parallels between the two; like the fact that in either work place I’m constantly sanitizing. In one job I’m growing bacteria, and in another I’m getting rid of it…and I honestly couldn’t tell you which one is which.

But, regarding juggling experiments at the Mayo Clinic, I’ve now got 3 active experiments going at the same time. One experiment is the drug titration, which determines the progress of cell proliferation. Another is the analysis of the RNA produced by each cell line. And the last is the western blot, to determine the protein production. All three steps will be the final determination of what our conclusion to the experiment will be. The end is approaching, and it’s exciting stuff.

The results from last week’s drug titration also came in, and it seems that there may be some off target effects coming from the inactive version of the drug. The inactive versions are meant to act as a control, having the same effect on the cells as those that have not been treated. However, when we reviewed the results, in some cases the cells treated with the inactive drug grew even more than those with no drug. This may hint that the drugs are having some unwanted side effects. But, we’ve decided to run the test again to make sure that our results are more certain.

Until then, thank you for reading!

Angela

Experiments: the endless cycle

Hello and welcome back!

The results are in! And they’re consistent—something I hadn’t realized was so important to experiments until now. After putting the U266 (the cell line that reacted to the drug, even though we didn’t expect it to) through a drug titration (an experiment to see at what concentration the cells are affected) it showed marked signs of inhibited growth when a drug concentration of 1 uM was added. This is good news, since it not only verifies that we are following the drug company’s protocol but also that I am not horrible at lab experiments.

Unfortunately for me, I must’ve done too well—because this week I had to extend the drug titration to all four cell lines. That means 16 different drug concentrations, 160 wells, and 800,000 cells. My poor thumbs.

We’ve also begun to look at the protein and RNA synthesis of the treated cell lines. The ASO’s are meant to decrease production of IRF4 by inhibiting its RNA production. So, we should expect to see a marked decrease in RNA and protein production in cell lines treated with the active drugs. It will also be interesting to see the results of the cell lines that did not react to the drug. It may give us a clearer insight as to why the drug has no effect. We will be using a western blot to determine protein production (something I’ve heard about in Biology class but have never tried). It’s bound to be interesting!

Until then, thank you for reading!

Angela

A solution

Hello and welcome back,

The results of the new experiment are in, and are filled with surprises. After conducting a MTS assay on all four cell lines, we were able to see the effect of the drug on cell proliferation by measuring the ATP produced. Though we did not know exactly what to expect, we did have the hypothesis that the U266 cell line would be resistant to the ASO’s because it does not produce MYC, and—we thought—would not be dependent on IRF4 because of the lack of MYC. However, we saw that the ATP production of U266 was cut in half when administered the active ASO’s. This hints that U266 may still be dependent on IRF4, despite a lack of MYC production. In contrast, the FR4, which does produce MYC, was not affected by the ASO’s at all—a very unexpected turn of events. We will be examining these cell lines in depth in the upcoming weeks to determine just why this is the case. Interestingly, the KMS11 and the XG7 reacted as expected.

But, now that we’ve answered the question of which cell lines are reacting, we’ve decided to test another direction of the experiment: determining at what concentration of the drug the cells become sensitive. So, we’ve set up drug titration experiment, whereby we’ve plated one cell line (in the same reduced concentration of 5K cells/100 ul) with varying concentrations of all the ASO drugs (active and inactive).

Of course, this sounds easy enough. But. It. Took. Forever. 40 wells, 16 stocks of drugs, and 96 pipette tips later, the plate is set to sit in the incubator for another 6 days before I can go through another 40 tips and a tube of cell titer-glo to see the results.

Until then, thank you for reading. Happy pipetting!

Angela