Evaluation of my Goals

Here are my goals:
I want to understand what I am doing in the lab at all times and to further my knowledge on anything that relates to biology and medicine.

I want to remain focused while I am setting up tests so that my data is accurate. 

I want to be able to form basic conclusions with the data that I get. 

I just wanted to go over them quickly:

I feel like I have definitely furthered my knowledge when it comes to medicine because of all of the talks that I've listened to. Whether it be Doctor Willey or the fellows I shadowed in the clinic, I know that I've increased my understanding about medicine. Unlike last year, I knew exactly what I was doing in the lab. I could troubleshoot without going to other people because I understood what went wrong from the data displays.

Based upon Xiaolu glancing at my data, I feel as though it is very comparable to her. In addition, I know that I was focused through the testing while I was at Winterim, and it seems to have paid off.

Because we ran out of chips, I didn't get to generate as much data as I would have liked. However, I do not want to base a large conclusion upon a few data points. I can see that there is a drop off in gene expression because of the siRNA, but I would want to run many more tests before I would form a conclusion.

Last Days at UTMC

Last Friday and Yesterday were kind of emotional days.

On Friday morning, we had to deal with the Inspector from the Department of Agriculture. Basically, I hid in the back setting up tests for two hours so that she wouldn't interrogate me. However, Erin did mention that I was apart of the lab staff, so the inspector demanded that I get high-level lab training. That to do that I needed to get registered in UTMC's database. The only problem is I had been recently deactivated. I needed to wait the weekend for the IT department to reactivate my account. I will do the trainings tomorrow morning.

For the rest of the time, I did the normal setting up tests and chips to get data. Overall, I didn't get nearly as many data points, but there wasn't much I could do about that. In my free time, I talked with Doctor Willey about what I'm doing for Winterim Fair, and I think that I have a pretty good board made up. Hopefully I'll see everyone there.

Also, on Monday morning, I brought in donuts as a thank you for all the members of the Lab. When I said my final goodbye, I got kind of emotional. I had working with a few of the members of the lab for the last two years. I had really gotten to know them and I knew that they had my back whenever I messed up. They all told me to come back and visit sometime. I most definitely will.

Throwback Thursday November 21st

When I mean Throwback Thursday, I mean I threw back to a time when I had chips and I could run them properly. Today I was able to run 2 chips (that's 24 samples) and get 6 data points (I had 5 at at the start of the day). All of my data made sense and seemed to be accurate based on Xiaolu's cursory glances. I felt really proud that I started to produce good data on my own. I can see why some people make research their lives' work. It was almost a sort of high when I saw that I had obtained good results. I can even imagine how ecstatic one must feel when they publish a noteworthy paper.

Speaking of which, I got to listen to Grand Rounds (a large weekly presentation accompanied with free pizza). The Dean of Medicine at UTMC, Dr. Hoover, talked about Renal Stenosis. He had found over the course of the last 15 years that the insertion of a stent into the closed renal artery (a costly and risky procedure) doesn't not have decreased risk of clinical incident (a major health problem like a heart attack or stroke) compared to treatment with new pharmaceuticals. He had even helped find a potential solution that would save kidneys and lives. He recently published his findings in a high level medical journal and praise was coming in. At the end, he cried and was greeted with a large round of applause, which he obviously deserved. But the thing that stuck out most for me was that he had spent 15 years of his life, many of those years away from his family, in order to obtain this conclusion. As amazing as he must of felt, I don't know if I would find it worth it to sacrifice 15 some years of family life in order for an amazing paper or two. Just something for everyone, especially myself, to think about.

I didn't run any tests in the afternoon because the National Institute of Health is coming in tomorrow to inspect Dr. Willey's Lab. Every one in our lab, including Dr. Willey, cleaned up in the lab area. Hopefully tomorrow goes well.

November 20th THAD THE SHADOW

This morning was the typical morning. I ran some PCR and froze the samples. To my extreme pleasure, the electrophoresis chips arrived in the mail. I almost screamed in joy. This means that I can get some data when I have time. However, I had planned to shadow Dr. Willey this afternoon since the chips hadn't arrived yet. All afternoon I followed Dr. Willey and his fellows at the Rupert Center.

Before I went to shadow him, I ate lunch with Dr. Willey. He told me that I wouldn't be seeing Lung Cancer patients today, but merely patients who had problems with their lungs. Doctor Willey is a Pulmonologist at the Critical Care and Sleep Medicine Clinic at the Rupert Center. He told me that they often lump both Critical Care and Sleep Medicine because they both intermingle with the lungs. Lots of the problems that deal with sleep are a result of some problem with the airways and usually with the lungs specifically. In addition, Critical Care usually involves people on ventilators, so they like to have Pulmonologists deal with those patients.

I followed one of his fellows, named Anthony, for the majority of the time. We saw 3 patients during our time and all three had severe lung problems. I noticed that they all shared some similar characteristics: GERD, COPD, Chronic Bronchitis , and Asthma, and nearly all resulted from extended tobacco abuse. Each time, Anthony would go in, get the history of the patient, and then report his findings to Doctor Willey. Doctor Willey would then talk to Anthony for a bit about what they thought was the best course of action. They all three of us would go and talk to the patient for the final time.

What I really enjoyed about shadowing Doctor Willey was seeing how doctors work. For the most part, people were talking the entire time. There was a constant flow of information from the patient to the fellow to the other doctors. It was amazing how fast their mouths were moving and all of the background information that needed to be known. A doctor has to be conversational, be extremely intelligent, know a tremendous amount of background information, and be able to solve problems very quickly. It seems like the perfect fit for me.

But at the same time, the research aspect of medicine requires nearly the same skill set. I could go into either clinical medicine or medical research... or potentially both, only time will tell. I hope to maybe shadow Doctor Willey again before I go to college.

Bigger Picture! IMPORTANT TO READ!

Background information: Currently, roughly 19% of people in America smoke cigarettes on a semi-regular basis. However, only 10% of people that smoke heavily get Lung Cancer. Lung Cancer kills the most people every year out of all cancers, mainly because it present asymptomatically until it is inoperable/untreatable. The reason that Lung Cancer occurs in a higher percent of smokers is that the inhalation of all the chemicals in the cigarettes causes many cells in the airways to die. The surviving cells then have to replicate more then they are supposed to, which leaves them at a higher risk for random mutations to occur in the DNA. Often these mutations are corrected by DNA repair enzymes, outside Macrophages who realize the cell is aberrant, or the death of the cell as a result of a mutation in a vital pathway. But since the amount of replications in the air way has been exponentially increased, the random mutations are more likely to add up to the point where a cell becomes cancerous. Thus, Lung Cancer is a fairly large problem in the smoking community, but not limited to just them.

The government allocates nearly $5 Billion a year for CT scans of smokers so that they can hopefully catch the disease in its incipient stages. Doctor Willey, a researcher of both lungs and cancer, sought a way to proactively test people for Lung Cancer. One of the major papers that Dr. Willey's lab produced in the last few years talked about a pattern seen with the levels of expression with certain antioxidant and DNA repair genes in airway epithelial cells (click here to read the paper). They found that there was a  "safe" zone in the amount of mRNA that would be transcribed from the cDNA that had been harvested from air way epithelial cells.

They concluded that for each of the 14 genes that the person does not fall into the "low prevalence" zone, the risk increases. 

Generally in the above graph the higher the number of genes that are out of the safe zone in the test (RTV) means a much higher chance of cancer. Most of the people with above 8 will develop cancer at sometime as they age.

Knowing the risk can allow for the people who haven't developed cancers to go through pre cancer therapy so that the cancer does not proliferate and kill them. This test was recently approved for usage across the country, and it will likely be used commonly by primary care physicians in the next year or so. Doctor Willey told me if they can cut the amount of CT scans they need for diagnosing cancer by just 20 percent, they've already saved the government $1 billion. This is because the biomarker tests are much cheaper and much more effective than the CT scans, saving lives and money.

In addition, another thing that is being looked at in the Willey Lab is a potentially easier method of extracting cells to test. Currently, they use cells that are harvested from Bronchoscopies, a process that takes a fair amount of time and effort to do. They're trying to find a way to get the same Lung Cancer test readings from different cell types, such as cheek cells or skin cells. This would make the Lung Cancer test even more convient and save even more money. 

So now you're probably wondering why this factors into what I'm doing. Well good question! I'm glad you're interested enough in what I'm doing to ask that. If you look above in the table with the 14 genes, you will see that CEBPG is one of the important genes. It actually has one of the smallest "safe" zones of the 14 genes. Based upon a paper that Dr. Willey's Lab produced in 2005, they know that CEBPG controls main of the important genes in air way cells, including many of the Antioxidants and DNA repair genes. All of the PCR and DNA chip electrophoresis has been centered around CEBPG. I'm helping Xiaolu and Doctor Willey with is seeing how treating cells with siRNA for CEBPG affects the expression of the other important lung genes, many of which are some of the other 13 genes in the Lung Cancer test. Depending on the results, it could be a treatment option or a new mechanism to be documented/researched. Hopefully, this has helped you understand more about the bigger picture around what I am doing this Winterim.

Lunch with Important People

Today was a pretty normal day for the most part. I reviewed the Real Time PCR results with Xiaolu first thing in the morning. I was slightly confused at first, but Xiaolu showed me how to get the number of targets produced per million ACTB by using exponential equations. I still don't understand it completely, but Xiaolu wants me to run a set of RT PCR before I finish Friday, so hopefully I will be able to explain it soon. 

After that, I started another round of PCR for a different cell line. Same old same old. I put the results in the freezer to run when we get chips. 

But then, I went to lunch out. Today, Mr. Boehm was supposed to meet with Dr. Willey and I to discuss how I was doing. However, Dr. Willey got called to listen and comment on a presentation at the last second. So all three of us went to listen to this presentation on Pneumonia and COPD (a lung disease). It was the first time in a while that I did not eat by myself. We ate sandwiches while the person talked over a special type of Pneumonia that results from complications with COPD. I was pretty interested by the topic, but I struggled to completely understand what was going on (mainly because I am still in high school). At the end of the presentation, Dr. Willey got up and answered more complex questions on the subject.

(Pictured is the building we ate lunch in)

After the presentation finished, we all sat and talked. Dr. Willey said I was doing a good job in the lab and at there were no complaints against me (yay). Mr. Boehm wanted to know more about why  research I was helping with mattered. This started a rather long and drawn out discussion about what's going on in his lab and Mr. Boehm eventually understood why what we were doing is important. I agree with him when he told me that he felt that my blog was lacking an explanation of the bigger picture. I will post again soon to clarify and explain why what I'm doing matters.

Also I'm planning on shadowing Dr. Willey in the lab tomorrow, so I will hopefully have a interesting post tomorrow. Stay tuned!

November 18th BUSY DAY

Today was easily the busiest day that I've had.
Even though the chips are still in the mail, I still wanted to do a lot of work. Doctor Willey and Xiaolu want to make an abstract before an upcoming conference, so there was an abundance of work in the lab.

(The list of all the work)

My job for this week is to get the ERCC5 and ERCC4 values for cDNAs from 3 different cells lines. I set up the test and ran the PCR. So that the PCRed samples do not deteriorate, I'm going to put them into the freezer. I currently have 25 samples that are waiting in the freezer for when the chips get back. I still have 24 more samples to make.

BUT I learned a really cool new test today called Real Time PCR. Xiaolu taught me that this test has 2 steps. For the first step, she puts in the different samples in a rack with 8 wells by 12 wells. She puts the same reagents in and for 18 cycles, it goes through PCR. During these cycles, the signals that the cDNA produces get amplified. Then, the samples are diluted to 1/10, 1/100, and 1/1000. I don't know the reasoning, but then  they undergo another 14 cycles of PCR. They then can determine how many molecule of the gene was produced per million ACTB (exactly the same thing I did with electrophoresis) based upon how much fluorescence is produced (it's produced by the probe finding its complementary sequence [check the wikipedia article it's kind of hard to explain]). Anyways, we get the same results, quicker and more accurately. Xiaolu and I are looking at how siCEBPG affects the production of MUC5B (another gene) in the cell lines.

Also, I talked to Xiaolu and the reason that they are looking siCEBPG knocked down cell lines is that they want to see (if there is) a mechanism involved when CEBPG regulates cell functions. I'll try to explain that more tomorrow.

My research hits a wall

Well... I had mentioned in my last post that I would run the results of my last PCR when the electrophoresis chips arrive... I did not anticipate that the chips wouldn't arrive until the middle of next week. As a result, I cannot get any more data for the time being.

With my down time, I wanted to learn more about what I was doing. I talked to Dr. Willey and asked him about the questions I had. The main thing that I had wondered was why they had chosen to study CEBPG. I had asked him this before and he had told me that it was a transcription factor. At the time, this had satisfied me. However, there are many transcription factors in cells, so now I wanted to know why CEBPG was special. He told me  CEBPG was believed to be the paramount transcription factor in regulating lung epithelium based on a paper Dr. Willey helped with in 2005 ( click here to read it). Basically, CEBPG controls important antioxidants and DNA repair genes.

So ultimately, I wanted to know what they were hypothesizing. Dr. Willey told me that they want to see how much the gene expression of genes regulated by CEBPG is effected when cells are treated by siRNA. Once they get data, they will hopefully be able to conclude how much the other genes are effected by using siRNA for CEBPG

Also, I never got to show where I was working. This is my little office area where I have been every day during Winterim:

I screwed up... Oh wait, no I didn't

Well today wasn't a normal day.
I started off the morning talking to Erin and Diego (a graduate student) about the electrophoresis chips. These chips are the most costly things that I use in the lab. Recently, Diego and I had been getting slightly messed up results with the chips. So Diego, Erin, and I all ran a chip together to make sure that our procedure in setting up the chip was proper. For the first time in a while, our data had no issues. However, there is only 1 chip in the entire lab left, so I cannot get new data from the PCR that I've run until we get more.

                                                                   (The holy last chip)

So, with the free time I had, I started calculating values and putting them into an excel sheet. When I looked at the data I had, I got a little worried. The cell line treated with siRNA was only 400 less than th control (treated with nothing) cell line, while the cell line treated with only the Dharm was nearly 100 higher than both of the other values. I went to Erin to see what I had done wrong.

She told me I hadn't done anything wrong. As I walked into her office, I realized that I left out a factor of ten in my control value. This made sense as the siRNA should silence the gene expression by over 70% (which in the case it did). Two of my data points made sense, but the Dharm sample still confused me. This was a result of my own lack of understanding.

Erin told me that when they use siRNA, they use the Dharm to get the siRNA into the cell. The cell membrane is hydrophobic, and the siRNA is hydrophilic, so the cell membrane (neutrally charged) does not want to let the siRNA (negatively charged) into the cell easily. The Dharm effect allows for the siRNA to pass into the cell through endocytosis by using liposomes. So all the Dharm-treated-only cells had were liposomes inside them. Erin then told me that the liposomes affect gene expression in both directions (higher and lower). As long as my data differences weren't too drastic (over a 1000 fold difference) that my data was still fine. Thus, I finally have data on all 3 of the cDNAs. I will start getting more data for CEBPG and for another gene, ERCC5, when we get more electrophoresis chips.

November 13 Getting Better

In the lab, today was another routine day. In the morning, I prepared my samples and ran my PCR. For the afternoon, I ran my PCRed samples through the electrophoresis chip and got some less than perfect results. This came as a result of problems with the ladder.

Time for some explanation. The electrophoresis machine measures how long it takes the sample to pass through the gel matrix. However, before the sample runs, the "ladder" runs through electrophoresis. The ladder is a mixture of molecules that vary in length. The machine and the ladder are produced by the same company, so the sizes of the molecules are already known before they're run. Since they know the 50 basepair (unit of measurement for DNAs and RNAs) group of molecules take  roughly 43 seconds, they can conclude the unknown length of the sample based upon how long it takes.

The problem was that my leader was off, so the sizes that the machine was given weren't accurate. As a result, I can't use this data. However, there is a bright side. I saved my samples and I can run them again (likely tomorrow because indeed to run some with Xiaolu's samples that aren't ready yet).

But more importantly, since I've gotten all 3 cDNAs into a good ratio with the competitor molecules, I should hopefully start cranking out data much more rapidly now. I will start running larger (12 samples per test) tomorrow.

During my down time, I looked through the paper that Erin sent me. I can't really explain exactly how they did it, but Tom and his colleagues found parameters that allow for the whole transcriptome sequencing to be more reproducible across different labs and for it to be done in extremely less (reduced roughly 10,000-fold) number of reads. I'll try to understand it more so that I can tell more about it.

Pretty Cool Day

Today was a pretty normal day in terms of running tests. I'm looking at the CEBPG for the final cDNA sample that I need in the preliminary testing.  Before, I had problems balancing the competitive template to native template, so I couldn't get accurate readings. This time, the peaks were within a 1:10 ratio, so I could get a number of CEBPGs per million ACTB (the standard unit across the tests). I encountered some problems with the machine and I also couldn't find supplies, so I ran out of time to check with Xiaolu. I will do this the tomorrow.

Also, I talked to Erin and Xiaolu, I will also run the tests for ERCC5 (another gene in the 14 gene biomarker) with the cDNAs that I have been using.

But, I learned some really cool stuff today. Last year, I worked with Tom Blomquist, another research in the Willey Lab. While I was gone, he helped make a new type of testing (with the help of nearly all of the other workers in the lab) that will hopefully help massively in the field. I just started reading the ninty-four page paper today, but I can give the gist of what they've developed. This paper actually hasn't been released, so think of this as your insider access into the scientific world.

A new technology called Whole Transcriptome Sequencing allows for the amounts of RNA (all types tRNA, mRNA, rRNA, siRNA, etc) in the cell at one moment to be measured. However, this method is costly and yield complex data that is often hard to interpret. Thus, Tom and the other contributors to the paper came up with a test, called Standardized RNA sequencing (Starseq for short), that involves running PCR with multiple primers and differing competitive molecules. This test allows for nearly all of the RNA values in the cell to be discovered by one (hopefully cheaper soon) test. This drastically cuts the time needed for people doing research to get their data. To quote Tom: "It's pretty friggin' sweet" 

This is what I've gleaned with the time I've had to read over the paper. Reading it is slow and slightly challenging for me, so I ask Erin when I have questions. Anyways, this test being developed would increase the reproducibility of findings in different labs while also cutting costs. I'll continue to read and I'll post more about what I learn about in the paper. Start tuned for some pretty stellar science.

Notes from 11/11

Happy Veterans' Day Everyone!
Well today I looked at a presentation that Doctor Willey sent to me; here are my notes about it:

So back in the early 20th century, a biologist by the name of Theodor Boveri hypothesized (and later proved) that all cancers started as one cell. A single cell that accumulates mutations in specific pathways will grow rampantly since it genes are "telling" it to keep dividing and to make more of the cell. The proliferation of cells then causes the classic, wretched symptoms of cancer. The pathways that are affected differ from cancer to cancer and from person to person. A doctor may find the chemotherapeutic drug that's being used might be completely ineffective against a certain type of cancer only after the patient has been on it for a time period. Thus, individualized medicine needs to be developed. Researchers need to develop more accurate biomarkers which can tell which treatment is optimal for each patient so that lives and dollars can be saved.

Some interesting tidbits:

• Lung Cancer remains the top killer among cancers according to the 2013 American Cancer Society Statistics.
• Lung Cancer kills more than Breast, Prostate, and Colon Cancer combined
• Ohio has one of the highest Lung Cancer death rates in the USA, and Northwestern Ohio has the highest death rates in Ohio.

Problems with developing new diagnostic methods:

• Current testing needs to be made less invasive.
• Small needles used for collected samples yield small amounts of samples
• Methods are not sensitive enough

Doctor Willey's lab has developed a 14 gene biomarker test, but it still needs refinement and approval before it becomes operational. This is just a bit of information for those who don't know exactly what I'm working on. Hopefully, more info is coming, so stay tuned!

Days "off"

Viewers,
You may be concerned that I did not post about my Winterim on Friday. You probably are waiting to here what I did on Friday. Well, I went to UT's main campus for the Junior Achievement of Northwestern Ohio Challenge. I played a business simulator in an attempt to win scholarship money. Though I did not win, I still did learn about business.
I will soon return to posting about my Independent Study.

HOWEVER. Tomorrow is Veterans' Day. UTMC is closed, but Dr. Willey will still be there seeing patients. I won't be going in because the other people in the lab will not be there, so I won't be able to get the materials I need or to check what I am doing.

NOT SO FAST. I'm not taking a day off though. I'm planning on going over a presentation that Doctor Willey sent me on Friday. I will post both the information I learn and the questions I have (which I will also send to Doctor Willey), so that I still further my knowledge about medicine.

Day 4 November 7

I mentioned in a early post what my morning consisted of. Doctor Willey sat me down and I learned more about what is going on in Medicine (see my other post from today for details). After he left, I went back to running the tests that I had set up yesterday. I had finally gotten everything balanced so I could actually possibly get some usable data this time.

AND TO MY SURPRISE, NOTHING WENT WRONG AND I GOT MY FIRST USABLE DATA. I could actually compare the 2 samples of cDNA I used  since all of the ratios were within the 1:10 to 10:1 guideline. The siRNA treated cDNA had a lot less amount of CEBPG produced for a million ACTB than the cDNA that hadn't been treated with siRNA. This makes sense because the siRNA should interfere with the amount of the gene produced. Thus, the one sample I got would support that siRNA can cut down the amount of the mRNA produced. However, they're looking at how much production is knocked down, so I will run the tests again Monday and start doing other samples. Should be fun to see what percentage the production gets knocked down by and if Xiaolu and I get similar percentages. I won't be posting tomorrow, so be sure to check in on Monday.

Lecturing

One of the ways I'm trying to make this year's Winterim different from last year's is by talking to Dr. Willey about what's going on in the field as much as I can. I came in roughly 45 minutes earlier than the other lab workers so that I could have him teach me.

First of all, I would like to say that Dr. Willey is a one of a kind man. He's extremely busy for this month, but he still came in early to talk with me. When you talk to him in his office, it's slightly intimidating because he's so poised and intelligent, but he attempts to make things informal as possible. He doesn't act arrogantly towards me even though I pale in comparison in terms of knowledge and experience. Doctor Willey is the kind of sincere professional I hope to become no matter what career path I choose.

So he started lecturing me about the "future of medicine". When I say lectured, it wasn't a boring monotone info session, it was more like a conversation between us. Even though we both knew the basics of cancer, he started with the basic knowledge of cancer before moving to the more advanced ideas. I had remembered that all cancers started as one cell who's regulatory functions get turned off, but he told me something very interesting: all cancers are different. The old idea was that each cancer in the body was just the same thing growing at different places and rates. Instead, they've now realized that each cancer is caused by disrupting different groups of  genes in a each cell for every person. Naturally should mean that every cancer (therefore person) should be treated with a completely different treatment regiment, but that would be way to costly to make/use. What people are now trying to develop are Biomarkers that could tell which type of pathways had been mutated in cancer cells;then, they could use medicines that they know are effective in dealing with cancers caused by a mutation in a specific group of genes. This would save lives and cut costs. Needless to say, I found this a very compelling notion.

Sadly Dr. Willey had to scurry on to a patient, but he assure me that we could meet other times. I'm looking forward to being taught by him again next Monday morning.

SMART goal revisions and other answers

1. I want to understand what I am doing in the lab at all times and to further my knowledge on anything that relates to biology and medicine.  I want to know what I am doing, how I am doing it, and why I am doing things in the lab. This means that I have a solid understanding of whats going on and that I'll be able to trouble shoot when problems arise. I plan on listening to other people's projects and exploring other abstracts to learn new information. I'll know I've accomplished my goal because I will have new information that I present and I'll able to explain exactly what I did in the lab at winterim fair.  This is a relevant goal for me because I want to go into microbiology and because I think I might go into this career path.

2. I want to remain focused while I am setting up tests so that my data is accurate. Last year, I know I was sloppier with my tests but I was new and was dealing with new concepts.  I want to reduce the number of careless mistakes I make during experiments. This year, I'm working more independently, so I want to be able work better without needing supervision. Measurable, we can see if my data has error or mistakes that were my own fault because it will yield bad data. In addition, I can compare my data with Xiaolu to see how accurate I am.

3. I want to be able to form basic conclusions with the data that I get. Last year I was more focused on learning about how everything worked. It's year I want to focus more on making tentative conclusions with my data if it is at all possible. I also want to know what the next step should be after making the conclusions. This means that I have to have both good analysis and good data. I would then present my conclusions at Winterim fair. Groundbreaking conclusions are not typically possible in 15 days, and I will only present a conclusion if it is at all possible.

In terms of my goals for communicating, I plan on posting at least once a day and telling about what i have done that day and what my thoughts were. My audience is primarily adults,  but also my fellow peers at MV who may look at my blog.

I plan to have a triboard with the charts and graphs for Winterim Fair

I know that I did a similar Winterim last year and I want to make a distinction between the two. Last year I was really just learning the learn things in microbiology and I got a little lab experience. Now I want to hone my skills in the lab and learn about some of the other things that people are doing in this field (both in Dr. Willey's Lab and outside of his lab) so I can if this is really something I could do all of my life.

Day 3 November 6th

Today was an interesting day.

In the morning, I did some PCR with ACTB (the reference molecule for nearly all of the tests in Dr. Willey's lab) and CEBPG (the gene that I'm focusing on). I felt like I did a better job of running the test and I hoped that my data would turn out well.  I waited for the tubes to go through PCR, then I loaded up the set into the electrophoresis chip. Everything was going according to plan, and I left for lunch so that I could look at results when I got back.

When I got back, I saw a blank screen. Normally, when I screw something up, the reading swill just be odd and off by a considerable margin. Not this time. Literally, I got no data. Naturally, I started getting worried; I thought I had broken the machine, which easily costs more than few month's tuition for MV. I ran to Erin (#2 in Doctor Willey's Lab) and asked her what happened.

Well, it turned out that the chip never ran. Apparently, every once in a while, the laser that is used for the electrophoresis doesn't get focused properly, so the entire process stops. Slightly annoyed that the machine didn't tell me so immediately, I needed only to load another chip because I had been wise enough not to throw my samples away (go me!)

Eventually, I got my data. Despite my struggles of getting back into the PCR groove, 3 of 8 samples were what they were supposed to be. 4 of them were wrong because we realized that my primer for the CEBPG (a necessary part of the PCR) wasn't working right, so it should work next time. However, 1 of the 8 was wrong and it's a good thing that it's wrong. One of the more interesting parts about the methodology of these tests is that I'm measuring the molecules relative to the known number of molecules that we insert. Now I have to balance these and get them into a ratio between 10:1 and 1:10 to make accurate conclusions. This means that I have to run more tests, which I'm excited for.

Anyways, if you saw Mr. Boehm's comments, you know I have some explaining to do. Check tomorrow to see my updated SMART goals and other new things for my Winterim.

Day 2 Frustration

Well, today was sort of an off day. I'm still shaking off the rust from last year's winterim and it showed. Today, I set up my first PCR experiment of this winterim. Even while I was doing it I felt like I was forgetting something. However, I continued and eventually tried to analyze my data.

Boy did I make a mistake. This whole process works because we can put a known number of competitor molecules in the PCR and then see the ratio of native (the strands we want) to the competitor molecules. However, based upon my data, I forgot to put the competitor in.

 

(how I felt)

But I realized that it was still just the second day, and that I was still performing tenfold better than how I was at this time last winterim. Plus, I was happy that I was able to troubleshoot and see my own errors. Tomorrow I hope to move past my blunder and start getting some data.

BUT! WAIT! THERE'S MORE!

Xiolu was showing me a different type of PCR: Real Time PCR.

Basically, what this method of getting data gets the same conclusion in a different fashion. It involves measuring the amount of fluorescence during each cycle of PCR. Although PCR occurs as a exponential function, the process eventually exhausts its products, so it reaches a limit. What they do it take the threshold at which the Native and the Competitor Templates reach their limits and then use this information to formulate a exponential expression that can be used to determine the ratio of CT to NT. And as I mentioned earlier, since we know the amount of Competitor, we can approximate the number of Natives made during PCR. This is basically the same result as what I've done and am currently doing, but it just can be used for different circumstances.

STILL NOT DONE. I also talked to Doctor Willey about the significance of the project I'm helping Xiolu. What he told is that since they are looking at how does CEBPG affect the transcription of the other genes important in keeping epithelial cells in the airways of the lung non-cancerous. Ultimately, they want to see how important it is in general. So hopefully I can help :)

SMART GOALS

Here are my SMART goals that I have come up after my first day. I may amend them, but I think they are solid:

1. To increase my knowledge about microbiology and to be able to effectively comminicate what I have learned.
2. To enhance the quality of my data collection by remaining vigilant throughout the duration of winterim.
3. To be honest and open with my data and be able to form conclusions with my data.






And just for reference, here are my SMART goals from last year:
"1. To actively engage in the lab activity that I am assigned so that I am beneficial to whomever I'm working with.
2. To further my understanding about genome sequencing and how it applies to lung cancer and to everyday life.
3. To behave courteously to all the personnel that I have interactions with at UTMC.
4. To effectively accomplish any of the tasks that I am given and report exactly what happened in the lab."
I hope to have similar goals for this year, but I won't know my specific project, so I don't want to make SMART goals ahead of it. On Monday, when I know what I'm doing, I will write new goals.

Nov 4 2013. Back to Basics

GREETINGS VISTORS

Well then....
I returned to the Lab of Doctor James C. Willey today. To my surprised I walked in to this sight:

The door is closed and locked if you can't tell.  What I hadn't mentioned last year was that November is the busiest month of the year for Dr. Willey since he sees patients in addition to his lab work. As a result I had to wait a few minutes for people to show up to even enter the lab.

The first thing that I was supposed to do this morning was get my project assigned to me, but since Doctor Willey had important conference to attend, I was alone for most of the morning. I kept myself entertained in productive fashion. I brought along my data and notes from last year and went over my entire project again. In addition, I looked up the old terms and procedures I needed to know for last year. That was my morning.

After lunch, Xiolu, one of the graduate students working with Doctor Willey, allowed for me to run some of her tests so that I could shake some rust off. I ran some PCR and loads some chips (the majority of the lab stuff I did last year) and it all was second nature. Hopefully I don't screw up her data.

When Doctor Willey returned, he said that I would be working with and under Xiolu for my winterim. She's pretty cool. No like actually she's really sweet and has been nice to me all of the time that I've been at the Willey lab. What she's currently studying is mRNA transcription rates in epithelial cells treated with siRNA. I probably lost a lot of you when I said that.

Breakdown: siRNA are Small Interfering RNA that interfere (who would have guessed) with the transcription of proteins More info here :). So basically, these cells (that are harvested from the cells that line your lungs) have been had some genes silenced so that others can be studied more intensively. The gene that we will be looking at is CEBPG,  a transcription factor, which is important in the testing that Doctor Willey is looking to implement. 

Anyways, even though it was kind of a boring morning, my biological adventure is starting to take shape again. I hope you all follow and enjoy my posts.

WELCOME BACK EVERYONE

I'm going to do another independent study where I continue my research from last year. I currently don't have any SMART goals because I still need to meet with Doctor Willey about what precisely my project will be.