methionine
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Cell Lines

Postby methionine » Tue Oct 30, 2007 2:14 pm

I am writing up a research proposal, and my project uses SK-N-BE cells (a type of neuronal cell). Is there anywhere I can learn more about this kind of cell... any sort of database or website that has information about different cell lines, etc.? Thank you!
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methionine
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Postby methionine » Tue Oct 30, 2007 4:23 pm

... agh, alright, nevermind that, I did a lengthy Google search and came up with some information. But I still have another question...
Could anybody explain to me how running a gel shows how genes are expressed (I'm only used to using the usual example - using, say, a plasmid and restriction enzymes, etc)? What I'm trying to say is that... my project is basically growing cells with different "conditions" (i.e. some have genes knocked down, others are normal, some are transfected to overexpress genes). So far, I've done RNA isolation, then amplified it through ReverseTranscription-PCR, using primers specific to certain genes I want to test. I'm basically trying to see if a cassette exon is included or not within those specific genes (as in... whether that specific "condition" my cells were grown in enhance exon inclusion or exclusion). What I don't understand is how it is possible to get two bands. I understand that either the cassette exon is either included or excluded, but I don't understand how both situations could be present in the same cells at once. I am supposed to be examining the ratios of the "strength" of the bands... but before this I had expected that I would only see one band: if it was longer, it would mean that the condition promoted cassette exon inclusion, and if shorter, it would imply that the condition in the cell suppressed cassette exon inclusion.

What concepts/ideas do I have down wrong? I understand if my explanation of my project was not clear enough... but if anybody could help me, I would really appreciate it. Thanks!!
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_Lisa_
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Postby _Lisa_ » Tue Oct 30, 2007 10:15 pm

methionine:

It is a bit hard to understand your proposed topic. What is the purpose of your experiment, your question?

I recently did a project myself running gels for a paternity testing simulation, so I hope that I can help you out.

-Lisa

methionine
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Postby methionine » Wed Oct 31, 2007 3:06 pm

How can you measure gene expression just by running a gel? Just... how does it work?
So far, the steps in my project:
- cell culture
- rna isolation
- ReverseTranscription - PCR
- run PCR products on agarose gel, observe/compare strength of bands... somehow this is supposed to determine .. gene expression levels or something like that. I'm honestly unsure.
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_Lisa_
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Postby _Lisa_ » Wed Oct 31, 2007 6:17 pm

Running a gel will tell you several different things. First, in order to understand what you want to find, you need to understand what running a gel will do. After cutting your DNA with a restriction enzyme and adding the other necessary components, the DNA is inserted into the agarose gel's wells and after a buffer is inserted, the electricity is applied. After about 45 minutes, the process is complete. Since DNA has a negative charge, it will repel itself from the negative pole and be attracted to the positive pole. The reason why the DNA travels different distances is because the smaller fragments move faster. This can tell you several different things. It can tell you what the sequence is of the DNA (if you know what sequence the restriction enzyme cuts at), it can tell you what is similar, what is different, and there are many other possibilities.

May I ask where you got your procedure and your project idea from? Perhaps there is some confusion in the communication of the project procedure.
-Lisa

methionine
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Postby methionine » Wed Oct 31, 2007 8:15 pm

... no, I'm not using any restriction enzymes....
I'm doing something similar to this:
http://nar.oxfordjournals.org/cgi/conte ... m485v1#B31
... except I'm not doing the first bioinformatics part because I already know which targets I want to test.
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ChrisG
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Postby ChrisG » Thu Nov 01, 2007 9:55 am

Hi Methionine,
I'm glad to see that things are moving along with your project. I recall that you were doing this work with a mentor in a research laboratory:
http://www.sciencebuddies.com/mentoring ... ght=#11374
Have you asked these questions to your mentor? Because they are more familiar with the planned procedures, they will probably be better able to understand and answer your question.

In the reference that you gave, Das et al., I don't see any discussion of gels. There is just the comment: "The identity of PCR products was confirmed by DNA sequence analysis." There are many methods for DNA sequencing, some of which involve gels and are discussed here: http://en.wikipedia.org/wiki/DNA_sequencing
I hope that is relevant and helpful.

methionine
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Postby methionine » Thu Nov 01, 2007 4:36 pm

from Daas et al:
A random subset of candidate muscle-enriched exons was selected for validation by RT–PCR


... I just wanted to know how RT-PCR results could be interpreted.
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methionine
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Postby methionine » Thu Nov 01, 2007 4:45 pm

I would ask my mentor, except I don't want to appear too lost. I mean, it's just that I feel like I'm supposed to know this already and I feel like I shouldn't bother my mentor / bombard him with questions. He doesn't exactly love explaining things, I think. This year I'm having a bit more trouble understanding things because I wasn't the one who actually designed the experiment.
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Louise
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Postby Louise » Thu Nov 01, 2007 4:58 pm

methionine wrote:I would ask my mentor, except I don't want to appear too lost. I mean, it's just that I feel like I'm supposed to know this already and I feel like I shouldn't bother my mentor / bombard him with questions. He doesn't exactly love explaining things, I think. This year I'm having a bit more trouble understanding things because I wasn't the one who actually designed the experiment.


Is there a grad student in the lab who could help? Or a tech? Even asking for a reference that would explain this topic in more detail would probably help you a lot...

At this point, you are working at a fairly sophisticated level. Unless someone here happens to do this type of research, you probably aren't going to get much help. I don't mean to discourage you from posting; the experts here work in a wide variety of areas and someone may be able to help. But, it might take longer to get an answer or you may not get an answer. I don't know anything about this topic, but I found these links. I hope they help:

This company has a lot of detailed information... obviously they push their products, but I think the overview is good.

http://www.ambion.com/techlib/basics/rtpcr/

This site has animation and links to other edu type resources.
http://www.bio.davidson.edu/Courses/immunology/Flash/RT_PCR.html

Louise

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Postby Louise » Thu Nov 01, 2007 5:01 pm

Also, the wikipedia entry on gene expression might help answer your question about RT-PCR and measuring gene expression:

http://en.wikipedia.org/wiki/Gene_expression

Louise

ChrisG
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Postby ChrisG » Thu Nov 01, 2007 9:24 pm

methionine wrote:I would ask my mentor, except I don't want to appear too lost. I mean, it's just that I feel like I'm supposed to know this already and I feel like I shouldn't bother my mentor / bombard him with questions. He doesn't exactly love explaining things, I think. This year I'm having a bit more trouble understanding things because I wasn't the one who actually designed the experiment.


I know what you mean. Of course you do not want to impose or be rude, but it is also important to remember that you are providing a very valuable service to your mentor, and it is in everyone's best interest that you understand why the experiment is designed this way, and how your procedures work. If your mentor seems to busy at the moment, can your arrange another time or, as Louise suggested, seek the help of another scientist in the lab? If all else fails, we will always do our best to help you here in this forum.

While it is important to be vigilant about understanding what you are doing, you probably do not need to worry right now about procedures that are weeks or months ahead in the project. Focus on learning as much as you can about your current procedures, and have faith that your mentor has devised a sensible experimental design, and that the procedure will become more clear when you are actually doing it. As my supervisor likes to say to me, "One insurmountable task at a time". :)

adance
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alternative splicing

Postby adance » Fri Nov 02, 2007 9:17 am

Hi Methionine,

I think I understand what you're asking...I did a similar experiment at the beginning of my phd thesis.

So what you've got now is a bunch of RNA fragments from RT-PCR. What this tells you is whether genes were transcribed, and in what form. Exons can be either included or excluded, as you said, and the piece will be longer if they're included.

If you see two bands on the gel...then you've got both options! There is some RNA with the exon, and some without. This is a natural thing to happen...it just means the cell is expressing more than one form. So if you compare the bands, you can say something like "most of the RNA has the exon, but some does not." Maybe both forms have a job in the cell.

In my experiment, there were even more than two bands--the exon had three different pieces that could be on or off! If you want to check out my paper as an example, here's the citation info (look at figure 1):
Traffic. 2004 Oct;5(10):798-813.
Regulation of myosin-VI targeting to endocytic compartments.
Dance AL, Miller M, Seragaki S, Aryal P, White B, Aschenbrenner L, Hasson T.

Please feel free to get back to me with more questions. But, I agree with what others have said--you should ask questions of the people you're working with too. Sometimes, professors are thinking at such a high level they just forget to explain the basic stuff. When I'm in a new situation, I always remind myself that it's better to ask the "dumb" questions straight away, then to stumble around on my own for a long time.

Hope this helps,
Amber
Amber Dance
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methionine
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Postby methionine » Fri Nov 02, 2007 11:01 am

Adance and all- thanks!
Yes, that was what I had suspected... now I realize that this is the reason why I'm comparing the "strength" of the bands to see if exon inclusion or exclusion was favored. But I never knew that expression of a gene could just be shown like that... I had always previously thought of it as a phenotype sort of thing (like... the cells will look funny, or behave in a certain way, etc., if they are expressing a certain gene); not something we could observe by simply running a gel and looking at the actual bands of DNA.

Haha. I feel sort of stupid whenever I have too many questions, because I usually feel the need to read everything closely by myself and then go into the lab understanding things. Plus, I usually like exceeding peoples' expectations (and I often do). But I don't feel like I'm helping my mentor... I think I'm probably more of a burden. For now, at least. Hopefully after I get confident enough to work alone I'll be a bit better.
People do not see the world as it is, they see it as they are.

adance
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Postby adance » Fri Nov 02, 2007 11:16 am

Methionine--actually, you make a really good point. By looking at the RNA on a gel, you haven't proven if the protein is really expressed, and whether it makes any difference to the phenotype. All you can say is that different versions of the gene are transcribed. This would be good to bring up with your adviser--think about ways you could make the next step of your project to analyze whether the two forms both become protein, and whether it makes a difference to the phenotype. Why do you think there might be two forms?

Nobody "works alone" in science (OK, I suppose there are a few antisocial freaks...) Researchers are constantly bouncing ideas off each other and asking each other's advice. At least, I hope you are in a lab with that kind of atmosphere.

When I was a grad student, sometimes I would struggle with a problem by myself for several weeks...then finally go to my adviser and find that she could help me.

Remember, your professor was willing to take on a HS student--to me that implies he/she is interested in mentoring a beginner. Already it sounds like you have made a contribution in finding that there are two splice forms. Many people enjoy explaining how science and research work (all the buddies on this website, for example). Definitely go to the grad students and postdocs around you, too.

It is awesome that you do the reading and try to understand things on your own first, and you can use that when you ask questions. Say something like, "I read these three papers, and I understand such-and-such part, but I'm still a little fuzzy on how..." They will be impressed with your effort, I think. (There are college students working in labs who don't go to that much trouble.)
Amber Dance

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