anaan
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Joined: Fri Apr 27, 2007 5:24 pm

RNAi confused

Postby anaan » Fri Apr 27, 2007 5:32 pm

For my science fair I was planning to work with animals sich as mice but when I found out that we were not allowed to work with any organism that has vertabrae but bacteria is allowed- i was a bit stumped again.

My project was to analyze the effect of RNAi when a virus was intorduced to the mice. Now that I cannot work with mice-does anyone have ideas as to what I can do?
Thanks!

drhamill
Former Expert
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Joined: Thu Aug 03, 2006 12:39 pm

Postby drhamill » Sat Apr 28, 2007 7:47 am

Anaan,
RNAi is a very interesting subject, and the importance of this relatively new discovery is highlighted in that the Nobel Prize in physiology and medicine this year went to two scientists who have really helped to elucidate what RNAi is and how it works. Wikipedia has an exellent, and well referenced, explanation of RNAi: http://en.wikipedia.org/wiki/RNA_interference. I encourage you to read this carefully before proceeding.

As for projects - you will see that the best understood systems for RNAi are multicellular eukaryotes (though bacteria have perhaps a sort of similar system). As you note, it would not be feasible to work with mice. Depending on what grade you are in, what facilities you have, how much time you have, and people who might be able to help you - you could look into the possibility of using C. elegans (a little worm) or arabidopsis (a plant) for your studies. (With all live organisms - even for bacteria, you should check with your teacher to see what the rules are.)

You might also consider a bioinformatics type project - where you mine data available on public databases for information on RNAi.

If you are not wedded to the idea of RNAi, but in general would like to use a modern molecular genetic tool(s), you might look at some of the topic ideas under Life Sciences (e.g. biotechnolgy and genomics) on the Science Buddies webpages.

If you firm up your ideas a bit and still would like help - please post back.

DH

anaan
Posts: 5
Joined: Fri Apr 27, 2007 5:24 pm

RNAi confused cont.

Postby anaan » Tue May 01, 2007 12:50 pm

I did some research and I think that I might be able to work with the worm C. elegans. The wikipedia article was very helpful-thank you. I was hoping to intoduce some sort of a virus to the worm then activating the RNAi.
I read somewhere that when a virus is indroduced to the cell, as a defence system the mRNA is cut into small base pairs siRNA by Dicer enzyme then unwound RISC there are is also degration so there will be no final protein.
I have to find a virus or something similar. By doing this, i hope to shut down that specific gene. As to what virus I should use- I don't know.

Any suggestions?
Does anyone have ideas that does not involve using a virus?
Thanks!

drhamill
Former Expert
Posts: 29
Joined: Thu Aug 03, 2006 12:39 pm

Postby drhamill » Tue May 01, 2007 2:49 pm

Hi Anaan,
While it's thought perhaps the mechanism of RNAi originally arose as a way to get rid of viruses (that contained dsRNA), this is not typically the way RNAi is done in labs. In C. elegans you need to inject (hard), soak in, or feed double-stranded RNA to the worms. While soaking and feeding are relatively simpler than injections, there are still some big challenges. You would need a well equipped molecular biology lab to prepare the RNA (or to grow cells with an expression vector from which RNA can be made). And / or you would have to spend $ to buy expensive reagents. You also need to learn to work with the worms.

Then there's the question of what you would "knock out". You could pick to knock out a muscle component, and perhaps see that the worms don't move properly. It's likely that any single gene you would chose to knock out has already been targeted by RNAi, and the results are available on the web. (http://www.wormbase.org)

You didn't mention what grade you were in nor what resources / help you would have, nor how much time you have for this project. I don't want to squelch your interest in / enthusiasm for a project with RNAi, but I must admit that I'm worried about how it could be done. Perhaps someone else will post in with ideas. If you do indeed plan to work in a molecular lab and you have a mentor who has some experience and is able to work with you, it's not out of the question. However, I would encourage you to think about other projects that also interest you. This one seems to be awfully challenging - lots of work for a scientific question that you haven't yet specified (apart from your interest in the technique).

DH

anaan
Posts: 5
Joined: Fri Apr 27, 2007 5:24 pm

Postby anaan » Tue May 01, 2007 3:08 pm

im in 10th and i have a college that is connected to my school so im preobably going to work there

drhamill
Former Expert
Posts: 29
Joined: Thu Aug 03, 2006 12:39 pm

Postby drhamill » Tue May 01, 2007 6:22 pm

OK.
Let me suggest a couple of references for you to keep researching RNAi and worm handling.
The first 10 pages of the following chapter from "Wormbook" are most useful for RNAi and it describes detailed protocols on preparing the dsRNA and administering it to worms:
http://www.wormbook.org/chapters/www_in ... netics.pdf

An overview of some of the other useful worm resources is provided in this source (also from wormbook):
http://www.wormbook.org/chapters/www_we ... egans.html

I hope after reading through these you have a better sense of what you want / can do.

DH

anaan
Posts: 5
Joined: Fri Apr 27, 2007 5:24 pm

Postby anaan » Wed May 02, 2007 4:15 pm

The methods for inducing the dsRNA into the C. elegans are complex but it all depends on my experiment and what gene i wish to target. I was thinking about using the mus-101 gene since it plays an important role in the C. elegans-for DNA replication, DNA damage repair and so on.

My science fair project will be analyzing how RNAi affects C. elegans when the mus-101 gene is targeted.
But I am confused as to why (according to the Reverse genetics paper that was generously supplied thank you =D) the reserchers used the embryo's in the RNAi soaking method.
For the purposes of my expermiment would i have to use the injection based method?

drhamill
Former Expert
Posts: 29
Joined: Thu Aug 03, 2006 12:39 pm

Postby drhamill » Thu May 03, 2007 9:39 am

You should not need to use the injection method. Basically, the dsRNA just has to get into the worms. One way to do this obviously, is by injecting it, but it also gets into worms by letting them eat bacteria containing your RNA of interest, or even soaking worms in RNA solution. Usually, if one method works, the others do too, but there are a few cases (seems to be gene-dependent), where injection seems to have a stronger effect than the other methods. (But as we discussed before, injections are hard to do!)

As for mus-101, it is an important gene. I'm pasting here a link to the information on this gene in the open database called "Wormbase". Half-way down the page you will see information on RNAi that's been done with this gene. 19 experiments have targeted this gene with defects ranging from nothing unusual observed (most common) to defects in locomotion.
http://www.wormbase.org/db/gene/gene?na ... class=Gene

For a science fair project, you will want to state a clear hypothesis that you can test, and I'm still not sure what you wish to "test". If your hypothesis is that mus-101 knock down worms will have problems with DNA repair, how would you test this? Would you try to inactivate this gene and see if these worms are more sensitive to other insults that are known to damage DNA? There's still quite a bit to be worked out here, but you're making good progress.

anaan
Posts: 5
Joined: Fri Apr 27, 2007 5:24 pm

Postby anaan » Thu May 03, 2007 3:10 pm

No, my hypothesis is not if mus-101 gene is knocked down using RNAi then it will cause defects in DNA repair. This of course would have been an interesting experiment. Maybe I could test if my hypothesis was correct by possibly exposing the cells of the C. elegans to something like UV rays then using Topoisomerases and quantifying the covalent complexes... But this will take too much time-and my time is limited. I cant really think of a hypothesis that is simple enough and will be beneficial to prove.

Maybe I can test if the mus-101 gene is knocked down then the C. elegans cannot move properly promoting faster death or slower growth? Or something else. I'm still thinking about it.

The link you sent me was helpful although it showed many of the things that the scientists did not test for. One thing that i did notice is that the mus-101 gene-when knocked down is respinsible for abnormal movement and paralysis.


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