Clonogenic Assay- How resistant are you?

The gene analysis is complete. There is a difference, but does it do?

That is where the clonogenic assay comes in. This might sound familiar and that's good because I have talked about them quite a bit. I never fully explained them (anyone else notice the trend... I'm working on it... I promise). The clonogenic assay is a method of testing radiorestance, or how resistant cells are to radiation. you start off by plating a number of cells on a 6-well plate (its a plastic rectangle with 6 wells just in case that was confusing) and then you irradiate them. For our experiment, we had six 6-well plate (for a total of 36 wells...Wow) and the irradiation treatments were from 0Gy - 10Gy. Duplicates of the three samples of the cell line were on each of the plates. This was completed for all the cell lines 4 times.

Here are the results:

Look at that all three cell lines are there this time. Those lines are cool... what do they do?

Solid question. Here's the answer (well almost): The lines indicate the radioresistance of the cells. On the y-axis, we have graphed the surviving factor, or the ratio of the colonies that have survived after treatment. This is the dependent variable for all you high-schoolers out there (that's me). On the x-axis, we graphed the radiation treatment (that's the independent variable). 

Some of you may be wondering how did all of these numbers were calculated. I appreciate your curiosity and applaud your interest, but to save confusion (and possibly effort) I will leave the explaining to the true experts. To find the full protocol, from irradiateng cells to interpreting data, click the link: http://www.nature.com/nprot/journal/v1/n5/full/nprot.2006.339.html. 

If you have read it, but still have questions, please ask. Now back to the show.

For 22Rv1, the cells that received the FD treatment exhibited greater radioresistance, meaning a greater number of colonies from that sample survived radiation. 

For DU145, there was no difference in radioresistance between the cells that received the two treatments. 

For PC3, the cells that received the SD treatment exhibited greater radioresistance. 

I don't mean to cut the party short, but that's it for the analysis of this data. Now before we begin figuring out what it all means there is one more thing to do. You guessed it! More data to analyze. 

Till next time, my friends!

Biostatistic Data- Bet you don't what that is!

Okay! We have compelted the CLPA analysis. Some genes were not expressed and some genes were expressed. Great, but what do we do now? Well, as I mentioned previously, to determine whether there is a significant difference in expression you must complete a biostatistic of your data.

What is that, you say? Here, I got you. Let me explain. Biostatisctics is the branch of statistics branch of statistics that handles the proper interpretation of data generated in health sciences such as biology and public health. Dr. Lacombe completed this analysis with the help of some software (thank you technology).

Here are the results:

Okay... 22Rv1... PC3... but wait one's missing. DU145, what happened?

Well, when running the biostatistic we were looking to see if any genes were deferentially expressed between the single and fractionated dose treatments. DU145, as seen by the lack of a graph, did not have any genes that were differentially expressed.

ATTENTION: This does not mean that DU145 is no longer an important part of the results. Not finding something has just as many implication as finding something.

Now back to the data.

As indicated by the graph above, in the 22Rv1 cell line there were four genes that were differentially expressed between the SD and FD. These genes were PARP1, CDKN1A, DDB2, and TRIB2.

For PC3, there were four genes that were differentially expressed between the two treatments. These genes were DEDD, RPA1, and MDM.

One thing to keep in mind: These that were differentially expressed between the two treatments were different between the cell lines. (Look back and check. It's pretty cool right.)

Here's the data! (well the first part of it)

The main event of this meet was the gene analysis. The differences in gene expression between the single dose and fractionated dose treatments is the main focus of this study.

The CLPA analysis and CE anlysis are the methods we used to analyze the genes. Here are the results:

I apologize for the lack of labels. Here's what you need to know:

• Starting from the left: the first three columns (0,SD, and FD) are 22Rv1
• The middle three (0, SD, and FD) are DU145
• The last three (0,SD, and FD) are PC3

That's great, but what does it mean?

Don't worry! I'm here to explain. 

The panel on the bottom is a legend. ( For everyone who thought of Drake when they read that sentence...same). The green (0) indicates that the gene is not expressed  and the red indicates that the gene is over expressed. 

For the three cell lines, there is a similar gene profile. The genes are expressed and the gradient expression is relatively similar. 

There are 6 genes that are not expressed in any of the prostate cancer cell lines. This is indicated by the light green on the graph. These genes are CD27, CEBPB, PRIM1, BCL11B, PTRCAP, and LAPTM5. 

There are three genes that are over-expressed in the three cell lines. This is indicated by the red or black on the graph. These three genes are RFC4, PARP1, and HIST1H3D. 

On the graph, there is also an indication of some differences in expression between the single dose and fractionated dose treatments for some genes. To determine if these differences are significant, we must do a biostastic. 

What's that you say?

Well stay tuned to find out!

Radiation Therapy (RT): What is it really?

As the amount of work in the lab dies down and the senior project comes to an end, I realize that I have never truly explained (at least in one post) the background of what I am doing and what I am truly doing. Since my posts have been mostly about my experience and day to day in the lab I am changing it up. These next few posts will be information on what I am doing and the results of what I have done.

To start off, a little background.

Radiation therapy. What is it?

Radiation therapy(RT) utilizes high doses of radiation to kill cancer cells and prevent them from spreading. It may be external or internal. External radiation is delivered through a linear accelerator while internal radiation is delivered from a radioactive substance a physician places in your body. This study focus on external radiation.

RT is extremely important to the treatment of cancer prostate patients (really all cancer patients, but since this study is on prostate cancer let's focus on that). 40-60% of prostate cancer patients receive RT as a treatment.

Conventional radiation is delivered in fractions of 1.8-2Gy per fraction. Patients will receive 35-40 fractions in total.

Recently, improvements in radiators and technology used in radiation therapy has allowed for the advent of hypofractionation such as stereotactic RT. This allows for higher doses and fewer fractions, meaning a patient will receive a dose of 7-10Gy per fraction and a total of only 5 fractions.

You might be wondering whether there really is a difference between these two therapies.

Well, there have been several clinical studies to evaluate the difference between the conventional and hypofractionated regimen on patients' outcomes. This includes things such as tumor resistance and overall survival.

Even though, the cellular responses have not been investigated and are still unknown.

This is where we come in. We meaning University of Arizona and me (for three months at least).

It's been so long!

Hello, everybody! (Imagine that was said in Gru's voice.)

It has been a while since I posted, which I apologize for, and a lot has happened, both in and out of the lab.

First off, I got college decisions. March was a month of Madness for college basketball players and seniors all over the country. Even though not all the decisions are what I hoped they would be I am happy and excited with the results. Also, CONGRATS to all the seniors! Even if you did not get into your number one, you all got into amazing places and should be proud! Wherever you go you will do amazing things!


Secondly, the lab has experienced some changes. The amount of work to be done in the cell culture room has greatly declined because Dr Lacombe has throw away the cells and begin with fresh cells for the new proteomic  assay. This has given me time to work on data analysis, which there has been plenty of. Luckily, with the help of my trusty friend excel, I was able to knock it all out in a matter of days. Two to be exact.

Lastly, there are far more problems to encounter in the lab than I realized. When doing labs in school, everything is though out and planned for us. The teacher and the students, if they are really paying attention, know what to expect. Very few real, lab-threatening mistakes are made. But in the real lab it's different and when mistakes are made the consequences are greater.

 For example, in the last clonogenic assay we made (  I will do a much more detailed post about it later) the cells did not get distributed the way hey should have been. There were too many cells in wells where there should have been almost none.

Another example: For the proteomic assay, the new cells have to be irradiated. Dr. Lacombe has been trying for the past two weeks to set up a time with the Mayo Clinic, but due to problems they are facing we are unable to get them irradiated as of now. This has a direct impact on the timeline that Dr. Lacombe had worked out to complete the experiments.

Finally, if anyone remembers, there are two parts to this study. One is determining the cellular response of three different cell lines to different radiation therapies and the second is building a radioresistant cell line to determine biomarkers of radiation. For the second study we have been using just the PC3 cell line and the cells have been very difficult to make radioresistant. The other day, while we were discussing the results of the experiments, Dr. lacombe informed me that he had read an article that said the PC3 cell line was known to be radioresisant already. No wonder it's been so difficult. Now, he is looking into possible changing the cell line he uses in the second study.

All in all, these past few weeks have been long, but exciting. Congrats again to everyone!

The one to explain it all (hopefully).

As I have been writing this blog and reading the comments, I have realized that my explanations have been lagging in some areas. I apologize for that. This post is dedicated to filling all of the gaps (or as many as I can) and answering all of the questions that have been asked.

The first question takes us all the way back to my first post. In this post I discussed two experiments for which I had completed he analysis but they were no longer viable. Since I never explained why, so I'll do that now. The experiment was no longer viable because the 0Gy plate for the clonogenic assay did not have any cells.

The second question was about training; the most exciting/ unexpected part of training and procedures that surprised me. The most exciting, or maybe best is a better word, is that you understand the why things are done a certain way, the labels on everything, and how to not retract a disease better because of training. The most unexpected part is that they had to tell us that we should not pipette with our mouths. This means do not suck the liquid (that has biohazardous cells/substances in it) through your mouth and put it in the place you want it to go. To me, at least, it seemed a fairly straightforward concept, but after reading the training further I realized why. Biosaftey and preventing lab acquired diseases, is a fairly recent phenomena. I do not remember the exact date, but it was within the 20th century that biosafety began to be addressed.

The third question was: what did I learn about the process of freezing cells by watching Dr. Lacombe? First and foremost, I learned about the full procedure. It is fairly quick and simple, but you have to act fast, which is the second thing I learned. When working with cells it is all about acting fast and making sure they are in the right conditions. To freeze cells, it is ideal to have a device that can bring the cells down to -80 degrees Celsius at a constant rate before placing them in nitrogen gas. Since the lab does not have such a device, they place the cells in a freezer set to -80 degrees Celsius for 2 hours and then place them in nitrogen gas.

The final question was: what it means to lyse a sample of cells and analyze the proteins. To lyse a cell is to breakdown the membrane of a cell. Once the membrane has been broken down, the proteins within the cell have been released. Since this is associated with the cartridge I do not have a very deatials explanation for how they will be analyzing the proteins, but generally when analyzing proteins you check for the amount of specific proteins expressed within the cell.

That is all. I apologize to all anyone who feels the gaps have not been filled. If you have further questions, please ask and I will do my best to answer them.

Over half way through.

The week of March 21st-March 25th marks the seventh week of the Senior Project. Less than half of the Trimester 3 is left. Five weeks sounds like a very long time, but time really flies, especially this year. This week I was only able to go to the lab for two days this week, but those to days were busy days.

On March 21st, I worked in the cell culture room. We were to create a clonogenic assay for DU145 and 22Rv1 and a cell viability assay for all three cell lines. Something I have not addressed before was the amount of time it takes. In the cell culture room there is no clock so there is no way of keeping track of time. When you are in one place for an extended period of time, your sense of time distorts. As I was doing the work, Dr. Lacombe informed me that it was already 6 pm. 6 pm? How could that have happened! After completing what little work was left, I left.  This was at 7 pm... luckily I made it home for bedtime.

On March 25th, I worked in the cell culture room and counted cells for a PC3 clonogenic assay. In the cell culture room, I changed the medium in the 96 well plate, trypsinized the DU145 flasks, and made tubes for CLPA analysis for DU145. In my next post I will share the data from the clonogenic assay and analyze the data. This time everything went much more smoothly and I am certainly more comfortable in the lab on my own.

After being in the lab and staying the few late nights, I suddenly have a greater understanding and deeper respect for everything that researchers do. Growing up, I spent a lot of time in the lab (well the lunch room but the lab room was right next door) because my mom is a researcher. As a child, I did not appreciate all the blood, sweat and tears that went research. Like any child, I was more impressed with all the technology, cells, and nitrogen gas than the people who did the actual research. Now that I have spent my share of time in the lab my perspective has certainly flipped.