Science Buddies: "Ask an Expert"

Hi, to whom may concern,
I was doing some background research on my science project, which is as follows, "Can the lengthening of Telomeres in roundworm species C. Elegans increase their lifespan and/or cancer vunerability?".
I am having some trouble in finding a proper method to A) increase the telomeres in the first place, and B.) Find a method to detect cancer in the roundworms. I read a short summary of a paper claiming that increase in the HRP protein somehow increases telomere length, but I have no idea how to and why HRP lengthens the caps. In addition to this problem, I do not know how to detect cancer in roundworms. All attempts at online research result in articles about human cancer detection.

Thanks,
-the desperately needy

Hi,

You may want to read this paper, which has a few comments on telomere length as it is related to lifetime, and telomerase, the enzyme which increases the length of telomeres:

http://www.new.dli.ernet.in/rawdatauplo ... 79_191.pdf

Here are some relevant articles on C. elegans and telomere length and lifespan:
http://www.pubmedcentral.nih.gov/articl ... id=1200426

http://www.plosgenetics.org/article/inf ... en.0010030

http://www.celldiv.com/content/2/1/3

http://www.jbc.org/cgi/reprint/M312011200v1.pdf

These book reviews could be of interest:

http://books.google.com/books?id=JjLWYK ... #PPA585,M1

http://books.google.com/books?id=ZEDzNr ... &ct=result

You have undertaken an ambitious project to say the least! I wish you every success with it. I hope these resources help a little. It looks to me like telomere length in C. elegans is only slightly related to ageing, which may be quite different to the situation in mamalian cells. ( I am definitely not expert in this subject area !!!)

Best regards,

Barrett Tomlinson

thank you so much for this helpful infomation! you are a real lifesaver!

I have another problem. I am not very sure about the process of starting with an organism and using gel electrophoresis to find its genomic sequence (I am interested in the measuring of how long the telomere bases at the ends of the chromosomes repeat, so this is necessary for a part of my experiment) Can somebody enlighten me in how you start with a living thing in a petri dish and end up with a piece of paper that has the ATGC markings? All research I have done so far only turns up results on how to prepare the sample and feed it into the gel chamber. The process of sequencing DNA is very imperative to the success of my project. Thanks.

Hi,

Can you tell us what sort of resources you have access to? Are you being mentored by someone at a university? With this sort of a project I would highly recommend that you find a mentor at a university who can help you. In order to sequence the DNA you will need lots of specialized equipment and may need to work with potentially harmful chemicals.

Here's a website from the Human Genome Project about how DNA is sequenced: http://seqcore.brcf.med.umich.edu/doc/e ... ncing.html . If you have any questions about any of that, let us know.

Hi,
I was looking at your first post and your interesting question B.) Find a method to detect cancer in the roundworms.
To be honest, I am not sure whether worms get "cancer" in nature but I do know the features of cells that lead us to call them cancer. In humans, the word cancer is used to describe a variety of different diseases that share some common features - the cells are growing out of control, they may have damage to their DNA, they may be resistant to the body's defense systems, they may have switched off the body's age controls, they may be resistant to drugs.
Sounds like you are interested in the age controls. You probably read that the telomere length is related to cell age. Simplistically, as the cell gets older the telomere gets shorter until at a certain point the telomere is so short that it generates a signal for cell to die and the replace it with a new cell. In cancer that process is broken and the old cell overrides the death signal and becomes cancer. Thats why lengthening the telomeres could make the worm vulnerable to cancer.
Going back to how to detect cancer, there a several ways depending on what feature you are interested in. SOme ways look at mutations or damage in the DNA. Other ways look at if the cells are overriding the death signals. The process of cell death signals is called apoptosis and involves, among other things, changes in the nucleus that can be measured with a stain and looking under a high powered microscope and changes in the DNA where it is chopped into pieces that can be seen on an electrophoresis gel. These types of experiments need the kind of equipment found in college level labs. So as Melissa said it would be great if you could find a mentor at a college or university. If thats not possible some of the helpful people here may be able to help you adapt your project so that its still related to your topic of interest but can be done with the resources you have.
Best of luck,
-Caroline

First, I would like to thank the respondents on this forum for this wonderful information. Without your help, I would probably have chosen a different topic a long time ago. However, I have several questions (again) about this topic.

So caroline, are you saying that through the use of electrophoresis, I can monitor changes in DNA and thus identify any change as cancer?

I am also considering to modify my experiment- slightly, depending on available resources. For example, do you think I discovered that because telomeres are part of the "cutting-edge" research topics of the decade, it incorporates the use of many advanced principles, such as fluorescent imaging, molecular manipulation, etc. You guys are right, I am in desperate need of a university- level lab and a mentor. The closest college with a good medical facility of the sort is Stanford University. Do you know which people I should send my "resume" to? I am considering Dr. Elizabeth Blackburn of and Dr. Steve Artandi as of now. In addition, I am not very sure how I can spark their interest. On the "How to Find a Mentor" site, it says to state that "you wish to conduct some research". Is there any particular question I should ask about?

Thank you for your enthusiasm about science, thats the reward that keeps us experts coming back! It really is great to see a student excited about a difficult but interesting topic.
Both of the researchers you mentioned are excellent and leaders in the field. Dr Blackburn is credited with discovering the importance of telomeres in cancer research. It can be tough getting into the lab as a high school student. Often at big medical schools like Stanford and UCSF they prefer to take students through a special program. There is a program at Stanford specifically for high school students but it runs over the summer (http://www.stanford.edu/group/cancerbio/summer.html). This would be great for you but is not going to help you right now. But i don't want to be all down - its definitely worth emailing professors directly but it may take time so maybe start another project so you don't get held up. It might be possible to do a bioinformatics type of project in cancer research or aging research that you can do from your computer. This would be good evidence to show a potential university mentor and they will see how serious you are about the work.
Back to finding potential mentors. If you are in the bay area there is a lot of choice - places like Santa Clara, Berkeley and USF, although not having a medical school and lots of nobel laureates, still do good research. So try browsing their websites too and looking for people with an interest in cancer research, telomeres, aging, apoptosis.
WHen you state your interests you should talk about the things you have been talking about here - telomeres, cancer and aging. Your actual research question is likely to change as you find out more. (and thats a good thing - when i was a PhD student my research question changed 3 or 4 times as i found out more details about my gene of interest and the end result was much better and kept me fired up more than if i had stuck with going down my original path)
Feel free to post drafts of your letters to the professors on the experts board if you want some help.
best of luck,
Caroline

-and yes, gel electrophoresis part of several ways to measure cellular changes due to cancer (see papers below). It is usually but not always coupled with PCR or sequencing and lately there are some high tech methods that people at the big medical schools use instead - but you don't have to use the fancy methods.
1. this paper is not in a prestigious journal but it is one that is free and shows the pictures of the gels using for measuring DNA laddering due to apoptosis. (and remember I mentioned before lack of apoptosis can be part of cancer)
http://vfu-www.vfu.cz/acta-vet/vol71/pdf/71_163.pdf
2. this is an excellent paper in a very good journal by Dr Blackburn and colleagues. This tells about how they use PCR to measure telomere length. In this paper it is done by a qualitative PCR machine because it can measure more accurately that measuring the intensity of a band on a gel.
http://www.pnas.org/content/101/49/17312.full.pdf

Thank you for the information on gel electrophoresis- it was very helpful and now I have a better understanding of how you start with chromosomal goo and end up with a nice printout of a genetic sequence.

Here is a rough draft of a letter I am planning to send to Dr. Elizabeth Blackburn. I managed to catch some of my mistakes that I had made in previous emails, such as not having enough background knowledge on the researcher's work and directly requesting mentorship. It would be great if you can have a quick look at it. Thank you for reviewing my draft, as it can ultimately decide whether I gain access to a lab or not.

Hi Doctor Blackburn,
My name is Eric Jang, and I am a student of Cupertino High School in the city of Cupertino. I understand that the researchers of the Blackburn Lab have made milestone contributions to the knowledge of telomeres and telomerase in cells. I watched a video of your lecture on this subject, and was very fascinated by the process which you used to determine that chronic stress indeed causes the shortening of telomeres and decreased telomerase activity (Telomere analysis of data from perceived stress tests results of many care-giving mothers showed that individuals with more chronic stress had shorter telomeres and less telomerase, thus leading to reduced ability of cells to divide and increased heart disease risk). I am also intrigued by the genetic manipulation of telomerase your lab group has conducted, especially the manipulated “scrambling” of a telomerase enzyme in Tetrahymena Thermophila protozoa to prevent them from renewing their telomeres and diminishing their immortality. I am interested in doing some research on this captivating subject, and would like to meet with you to talk more about your work in this field.

Sincerely,
Eric Jang
Cupertino High School


I think it sounds a little bit choppy, but am not sure how to reword my statements.

If it helps, here is a copy of a letter I sent to one of the postdoctoral fellows of the Artandi Lab a few weeks ago.
Hi Dr. Hsu

My name is Eric Jang, and I am a freshman student at Cupertino High School in Cupertino, California. I and my partner Chris Hsu are participating in the Synopsys Science Fair Challenge this year here in San Jose. While we were looking for potential research topics for our fair, we arrived upon the subject of telomeres, which we found to be very fascinating.
Chris and I have been particularly intrigued in the relationships between telomere length and the speed of senescence (Joeng KS, Song EJ, Lee KJ, Lee J 2004). Since you are a member of the Artandi Lab researching telomeres and have expertise in this unique area of science, we would like to ask for your help in this field by mentoring us in our research regarding the manipulation of telomere length. We would like to conduct an experiment to determine whether increase of telomere length in Caenorhabditis Elegans roundworms increases their lifespan. If feasible, we would also like to experiment whether the lengthening of telomeres in the roundworms is a tradeoff between aging and cancer rates.
We understand that the researchers of the Artandi Lab at Stanford University have already done research on telomeres, particularly concerning telomerase, cancer, stem cells, and senescence in eukaryotic cells. Since our desired experiment is quite complicated for students of our level, we require the education necessary to properly execute the experiment. We require much more knowledge of the structure and functions of telomeres as well as cancer detection and certain scientific procedures needed to gather data correctly in our experiment. We would like to meet with you to discuss our plan about telomeres. Your support will be greatly appreciated and very helpful to the development of our science fair project.

Thank you,
Eric Jang and Chris Hsu
Cupertino High school


(The reason why my former partner no longer is part of the letter is due to the fact that he decided to engage in a different field of science)

Hi Eric,
Your message sounds great. In my own career I have fielded some requests like this and I often have to send out requests to colleagues for assistance with peer-reviews. I can offer a couple of small suggestions. (1) I like the start of your second message where you begin by explaining who you and what you are doing. (2) I like the brevity of your first message. (3) I suggest making a simple & specific request at the end of the message, on a separate line to catch their eye. For example:

"Could you please let me know when would be a good time to call you to discuss a possible project on telomere lengthening in roundworms?"
(or whatever you'd like to discuss).

If you don't hear back within a few days, resend the message with an additional note at the top that you want to make sure that they received the message and you are still interested in speaking with them (very brief). If you still don't hear back, try again. The squeaky wheel gets the grease.

Good luck!
Chris

Thank you for your input on my essay, but I have a couple more questions. Here is a copy of a revised edition that I made. Essentially, it is the same except for the second to last sentence (I mentioned "experiment"- is that okay or too intrusive?), in addition to the separated closing statement. Also, I was thinking about informing Dr. Blackburn that I have done a lot of research already by myself, in case she doubted that a high school student could carry out an experiment of this type (I feel like if a highly educated proffessor of this field looked at the email, they would think that I lack enough knowledge about these things). It would be great if it had one more review just to make sure its okay before I send my email.

Hi Doctor Blackburn,

My name is Eric Jang, and I am a student of Cupertino High School in the city of Cupertino. I understand that the researchers of the Blackburn Lab have made milestone contributions to the knowledge of telomeres and telomerase in cells. I watched a video of your lecture on this subject, and was very fascinated by the process which you used to determine that chronic stress indeed causes the shortening of telomeres and decreased telomerase activity (Telomere analysis of data from perceived stress tests results of many care-giving mothers showed that individuals with more chronic stress had shorter telomeres and less telomerase, thus leading to reduced ability of cells to divide and increased heart disease risk). I am also intrigued by the genetic manipulation of telomerase your lab group has conducted, especially the manipulated “scrambling” of a telomerase enzyme in Tetrahymena Thermophila protozoa to prevent them from renewing their telomeres and diminishing their immortality. I am interested in doing some research and experiments on this captivating subject, and would like to meet with you to talk more about your work in this field.

Could you please let me know when would be a good time to discuss some questions about telomeres and telomerase?

Sincerely,
Eric Jang
Cupertino High School

Hi Eric!
I agree, I really like your letter. It's brief, yet it also pinpoints exactly what you want to convey. I would suggest that perhaps you could include some questions you might have about the topic right before the last sentence just to let the professors know that you have really been thinking about this topic and that you're really interested in it. Hope this helps, good luck!

Thanks! I completely agree with you about asking on one or two questions to show my interest in the topic.

Here is a question that I thought will show my interest and background research in telomeres and telomerase, especially on her work. However, it feels a little bit lengthy, and it defeats the purpose of a consice letter. Is there any way I can shorten it?

question (addressed to Dr. Blackburn) : Upon reading your published journal article on accelerated telomere shortening, it involved 58 (19 control and 39 "caregiving")mothers alike in every way possible, and the blood samples were collected on the first 7 days of the menstrual cycle (to produced the most unbiased data). Although it is true that both males and females can experience stress, it appears to me that there is more room for error in female subjects, as perhaps the genetics of a disabled child could have influenced the telomerase activity as a result of a genetic error during pregnancy. In addition, phases of the menstrual cycle may naturally result in different levels of telomerase activity. Males seem to me as potentially better test subjects as they are not subject to these factors. I feel like chronic stress produced by other outside variables, such as overdue taxes, divorce, or upcoming finals are more stable forms of stress-inducing vectors.

Hi Eric!
That's true, it is a bit lengthy although it's a really great idea. I think you can shorten it by simply stating:
After reading your journal article on accelerated telomere shortening observed in 58 mothers, it appears to me that there is more room for error in female subjects, although it is true that both males and females can experience stress. Could the genetics of a disabled child could have influenced the telomerase activity as a result of a genetic error during pregnancy? In addition, perhaps the phases of the menstrual cycle may naturally result in different levels of telomerase activity. Could males prove to be better test subjects as they are not subject to these factors? I also feel that chronic stress can be produced by other outside variables, including overdue taxes, divorce, or upcoming finals.

Hope this helps, good luck!

Thank you for the feedback! Now I am sure that my letter exudes confidence as well as knowledge. Also, thank you to the rest of the experts and moderators that helped me with the letter- having a mentor certainly beats going without. I will send my final email tommorow- wish me luck!

Here is the final thing, just for the record. (Note:The letter is still open to comments, questions, etc.)

Hi Doctor Blackburn,

My name is Eric Jang, and I am a student of Cupertino High School in the city of Cupertino. I understand that the researchers of the Blackburn Lab have made milestone contributions to the knowledge of telomeres and telomerase in cells. I watched a video of your lecture on this subject, and was intrigued by the genetic manipulation of telomerase your lab group has conducted, especially the manipulated “scrambling” of a telomerase RNA component in Tetrahymena Thermophila protozoa to prevent them from renewing their telomeres and diminishing their immortality. I am also very fascinated by the process which you used to determine that chronic stress indeed causes the shortening of telomeres and decreased telomerase activity (Telomere analysis of data from perceived stress tests results of many care-giving mothers showed that individuals with more chronic stress had shorter telomeres and less telomerase, thus leading to reduced ability of cells to divide and increased heart disease risk).
I am interested in doing some research and experiments on this captivating subject, and would like to meet with you to talk more about your work in this field. After reading your journal article on accelerated telomere shortening observed in 58 mothers, it appears to me that there is more room for error in female subjects, although it is true that both males and females can experience stress. Could the genetics of a disabled child influence the telomerase activity as a result of a genetic error during pregnancy? In addition, perhaps the phases of the menstrual cycle may naturally result in different levels of telomerase activity. Would males prove to be better test subjects as they are not subject to these factors? I also feel that chronic stress can be produced by other outside variables, including divorce or work stress.

Could you please let me know when would be a good time to discuss some questions about telomeres and telomerase?

Sincerely,
Eric Jang
Cupertino High School

Hi Eric,
That is looking great. Good luck. I hope you get a positive reply.
Keep up the good work,
Caroline

From my knowledge of biology, I believe my AP biology teacher said there were current studies on how the lengthening of telomeres that could potentially help us extend our lives as well.

Also, to my knowledge, I believe that if we lengthen telomeres at an early start it may just increase the length of life because as we grow older our telomeres get shorter and shorter as they slowly get cut off. There is no definitive study that shows that telomere lengthening may be the key to life lengthening but there seems to be potential in the source. Originally during DNA replication telomerase is the enzyme that increases the length of our telomeres, so information on that could also lead to more information on your topic.

Here is a site that directs you to studies on the different methods of telomere lengthening:
http://www.futurepundit.com/archives/000664.html

http://www.nature.com/ncb/journal/v9/n1 ... b1664.html (explains differences in telomere length in male and females)

Thanks for the cool info!


I have an additional technical thing that I have some issues with, concerning the T/S ratio.

I met with an expert in the area of biology, who currently works at Stanford. She told me that of all DNA assay methods, PCR is the fastest and cheapest way to conduct test- each only takes approximately 5 hours, and DNA samples can be frozen between experiments, which makes telomere assay via PCR probably the #1 candidate for measuring telomeres in my experiment. I have done a lot of research on telomere assay through PCR, of which is summarized along the lines of

"Mean telomere length and telomerase activity were measured quantitatively in PBMCs that were stored frozen at -80C. Telomere length values were measured from DNA by a quantitative PCR assay that determines the relative ration of telomere repeat copy number to single-copy gene number (T/S ratio)" (Blackburn 2003).

I am not very sure to what this means- could somebody please explain the T/S ration in a more "layman's" terms? I do understand the concept of PCR, which involves the mass-production of a certain gene through the use of taq polymerase and multiple cycles of heating/cooling. However, I do not see how telomere length can be "measured" in this way.

Hi Eric,

That's great you have talked to an expert at Stanford. This is my educated guess at what the T/S is about but check back with your expert.

The telomere is a series of repeats of the same sequence. The longer the telomere the more repeat units. The PCR is designed to amplify the unit sequence. So if you do a quantitative PCR you can measure the number of unit sequences, then you would divide by the number of chromosomes to figure out how many units per telomere and so how long they are. But how do you know how many chromosomes you started with? That's where you use the single-copy number gene. It is a control. It should only amplify once chromosome per round of PCR so gives you a way to measure how many chromosomes you started with. So the T/S is a way of finding the relative length.

Hope this helps,
-Caroline

Hi Caroline,

Thank you for your concise explanation. Not a single one out of the many journal articles I have read has explained the concept of PCR assay so well as you have

I have a couple questions though. In your response, you mentioned that "you use quantitative PCR to measure the unit sequences". I am not really sure what you mean by that, would you mind clarifying on what the difference is between quantitative PCR is and how it is used to measure unit sequences?

Also, I don't quite follow the concept of the single-copy number gene.

Sorry for the inconvinience



I have begun to flesh out my experiment. I have decided to do an experiment to find the increased telomere shortening in yeast when exposed to a stress vector. I have also figured the best way to conduct such an experiment was to have 3 groups: a control that is not affected, a test group that is exposed to an amount of the stress vector, and another group that is exposed to a larger amount of the stress vector. I have ruled out that temperature may not be a good idea because it can change the division rates of the yeast. I am not sure whether this may be important to the experiment, as telomeres are already known to progressively shorten with each division (thereby groups that have lower division rates will naturally have a longer average telomere length). I am considering sound to be a testable variable, although that requires the use of expensive soundproof containers and extremely loud speakers that must be kept on for days. Although sound may be a good independent variable, I am not sure if it is a good thing to test. Is there some other variable I can use in place of sound pressure that can induce some form of short-term physical damage while affecting cellular division rates as similar as possible, and at the same time, easy to apply?

Hi Eric,

Your questions are smart questions and not at all inconvenient.

Quantitative PCR uses DNA bases that have a fluorescent probe attached. The quantitative PCR machine can measure how much fluorescence was incorporated and so how many copies of the unit sequence were made.

The single copy gene is a gene that occurs only once in the genome. I guess generally we assume most genes occur only once in the genome but actually thats not the case, there are some that are duplicated many times and for some the number of duplications can vary between different people. To be a good control it needs to be a sequence that occurs only once in the genome.

Sounds like sound might be tough
Other types of stress that are used in yeast that i can think of are cold stress and nutrient stress but i would think these would also effect the rate of division. Change in pressure or oxygen would require more specialist equipment i think. Might it be possible to have more test groups and try 2 groups for heat and and 2 for cold stress assuming then that they have opposite effects on division rates but are equally "stressful"? I don't know too much about yeast though.
It would be worth doing a literature search to see if these kinds of experiments have been done before. There are lots of stress response experiments that have been done, perhaps some have a good stress protocol but their end goal was measuring something else other than telomeres. You could then adapt their protocol for your needs.

Best of luck,
Caroline

thank you for the clarification!

Application deadlines are coming up, so I am running out of time to submit my application. Working in a lab this year will probably not work out, so I will have to try to optimize my experiment so that it can be done at my high school. I have contacted SCCBEP, whom may be able to provide me with materials and resources. I need something that will be able to sequence the DNA so that I can measure telomeres- Which of the following kits would be best for me? Is it possible to compare telomere lengths by running a gel electrophoresis?

Here is the link to the SCCBEP resources website

http://www.science.sjsu.edu/SCCBEP/sche ... dules.html

Hi Eric,

The method we were discussing before - quantitative PCR - requires a special quantitative PCR machine (sometimes called a Taqman machine which is a brand name). Quantitative PCR machines are much more expensive than regular PCR machines so you wouldn't get one for a high school lab. Plus the reagents (the fluorescent bases and primers) are expensive. These types of things are not in the kits from SCCBEP.
In theory you could do regular PCR and try and quantify the products from the intensity of the band on a gel (You wouldn't need to do "sequencing" as you don't need to know the bases present in the telomeres just the length). But measuring the intensity of the band on a gel requires a special type of digital scanner/camera and software (which i didnt see listed in the kits) and is not all that accurate. Given the difference in accuracy between trying to measure intensity on a gel and a quantitative PCR, I would think it would be hard to see any differences with your experimental conditions, but i might be wrong. It would be worth talking to your expert at Stanford to see if she can help suggest some ways to modify your experiment.

I am having a hard time coming up with an idea to adapt your experiment to a high school lab and still keep the experiments central goal to be about telomeres. The only things i can think of involve doing a bioinformatics experiment about telomeres. I am kind of biased because bioinformatics is my area of interest - using computers to look at previously collected data and do molecular biology experiments without a lab.

Best of luck,
Caroline

Caroline, thanks for the consideration!

Although it isn't necessarily great that you had a hard time coming up with an idea to improve my project, I am very touched to hear that you spent time trying to figure out a way to make my experiment better/easier. My experiment, due to resource problems is stuck at the moment, but I am nonetheless glad that there are helping hands out there.

I have found an email exchange on the internet, of which I have posted a link below:
http://www.bio.net/bionet/mm/ageing/200 ... 04292.html
In that mail, I think it states that if you chop up the DNA with a restriction enzyme in the southern blot, it will not affect the telomeres anyway, and then that can be measured through gel electrophoresis by comparing the longest strands of DNA. Since the organisms I am experimenting with are the same species, any enzyme I use should leave the telomeres alone, and the lack of precision in separating the telomeres will not make much of a difference becasue the same will apply to the rest of the samples. I am not sure if this is true, but it is my presumption.

( I think the RSA/Hinf 1 enzymes are the restriction enzymes used for telomeres, as far as the link below says. http://cgn.pubhealth.ku.dk/upload/appli ... wborns.pdf )

However, you are correct that to measure the telomeres, I either need either PCR to amplify and then use a camera/scanner, or resort to southern blotting + probes. None of these are especially promising, as I either require expensive chemicals or hardware. There is an alternate method I am considering, called southern blotting. In normal Southern blotting, you basically run a gel of the DNA, and then you measure the intensity of a band by using a chromogenic dye or fluorescent probe. This is feasible to do with relatively simple equipment, save for it uses expensive chemicals. In addition, it requires many hours of waiting, in which I cannot be there 24/7 to replace paper towels, etc.

Caroline, I think the scanner+camera, although expensive, is the fastest and safest way to go. Is this link related to the kind of hardware you were talking about?
http://www.biocompare.com/Articles/Prod ... pment.html

Thanks,
Eric

I think I have found some good news. I was conducting another 2-hour internet search today and stumbled upon this website
http://www.bio.net/bionet/mm/plant-ed/2 ... 07518.html

I think the person is talking about how you can do analyze gels by using a program called ImageJ and a standard camera. I downloaded the program and looked at some screenshots on the web, and it appears to me that it indeed has the capacity to measure band lengths, and therefore compare average telomere lengths. However, being an open source program, there are hundreds of plugins and macros online that I have no idea what they do and how they work. Could somebody please teach me step by step what I have to do to use this program to analyze a gel? (I am no computer whiz).

The link to the program's website is here, if it helps
http://rsbweb.nih.gov/ij/index.html

I talked to my adult sponsor (my science teacher) and she says that in my experiment, to keep the temperatures in the yeast constant, I should use a heat source that does not produce UV radiation. I am not sure what kind I should use- an infrared heat lamp perhaps? would that produce UV light anyway?

Thanks!
Eric

Hi Eric,
Sorry its taken me so long to get back to you.
The things about taking an image of the gel and scanning and quantifying is that you need some kind of stain to measure the DNA bands. The most commonly used stain to visualize DNA is ethidium bromide, which is a carcinogen and i'm not sure what kind of approval you would need to use it. As long as you wear gloves and don't inhale it it should be fine but i don't know what the science fair rules are. Maybe your teacher knows. There are other newer types of stains which are less toxic but more expensive.

I'll try and take a look at ImageJ and see what I can figure out.

As for a heat source, in most incubators the heat source is in a separate compartment and the heat is conveyed by convection. Ordinary lightbulbs don't emit much UV and i guess could be used as a heat source. Might be worth searching old posts on these boards for answers to this Q.

Hope some of this is helpful,
Caroline

Thanks so much!

Yes, I am planning to use ethidium bromide for my experiment. I considered the alternative stain methods, but figured that it probably is too difficult to obtain SYBER-green or SYBER-safe staining methods. My teacher says ethidium bromide is okay, as long as she handles that chemical when it is being used. As for whether it is okay by the science fair, I will know if SRC approves the experiment.

I have run into a little problem, however. Today, I wanted to test how long yeast can ferment, just to assess what lifespan it has. I noticed that after about 5 hours or so, the yeast is starting to look a little unhealthy. I couldn't find any information on the lifespan of yeast on the internet, as the only results that showed up were about yeast infections and the cell cycle of yeast- none addressed how long yeast could keep dividing. This presents problems, as in my experiment, I run a gel on the same batch of yeast every two days to find telomere length. I may have to change my procedure or my model organism if yeast proves to be too short-lived.

Thanks for looking into the ImageJ software!

Eric

Hi Eric,
Sorry i didn't see this before.
I have to admit I haven't done much yeast cell culture but have done a lot of bacterial and mammalian cell culture and hope I can help (and i expect some other experts will chime in too!).
What conditions are you using? what kind of media are you growing in and what size flask? how much yeast are you putting in to start the culture? and how do they look unhealthy?
-Caroline

Hi Caroline

Actually, I have some good news. I couldn't find any information on their lifespan in my research, so I conducted a mini-experiment myself. Luckily, yeast is an exempt organism. Over the past two weeks, I ran a mini experiment with one culture of yeast suspended in a sugar solution. I added some sugar every day and warmed up the water in frequent intervals. I have determined that the yeast CAN live for the course of my experimentation cycle, which is at least 10 days.
Also, I figured how to use ImageJ to use densitometry and find the relative area of the bands, measured in pixels or however I would like it. I think the outline of instructions is here: http://www.lukemiller.org/journal/2007/ ... thout.html
I ran some tests with some sample gel images that I found on the internet, and it actually works pretty well.

Now all that remains is the problem of determining whether the telomeres are indeed the largest digested fragments after I run them through with the hindIII enzyme (its probably the only one I am able to get).