Advice needed about a science project

[Garret]

Well around mid-August I watched an episode of Nova that was showing a brief explanation of RNA interference and its application. So I decided to do a little research into and came to realize that this is something I would like to do for a science project. This is generally the time of year that I start a science project for science fair. The problem is, I’m in my last year of high school and up to this point we have not studied RNA in detail, so trying to figure out where to start and what to do has become pain. Genetics and the manipulation of it has always fascinated me, I’m somewhat of an advanced-academic-nerd so I’m willing to take up the challenge of learning and understanding the applications of RNAi or genetic engineering. Though I have some questions:

1.) I find the problem I have with every project is finding where to start and upon what angle to take. So where should I start? Should I take out some textbooks?

2.) I’m familiar with the petunia that was put through RNAi and eventually turned white rather than a deep purple when gene copies of key enzymes for flower pigmentation were introduced. What I don’t understand is how I could replicate this in the lab or something possibly simpler? If someone has a source of the procedures could someone inform about how to get them?

3.) What are some other fields of genetic engineering that have potential for study?

Thanks to those that help! And other comments would be greatly appreciated.

Regards,
Garret

 

[Abodunrin05]

Sorry, but i can't help you there. I am also planning to do a project involving genetics for my A2 (if you're not familiare with the british educational system, it's the last year of the A-level(Advaned level)) Biology. But when i find something, i'll make sure i'll inform you immediately.

 

[iansmith]

1.) I find the problem I have with every project is finding where to start and upon what angle to take. So where should I start? Should I take out some textbooks?

Usually the best start is to find a intro. to genetics book for university. However, RNAi has only been popular in the last few years, so it might not be included in the book.

Another source of information is the web:
http://www.ambion.com/techlib/hottopics/rnai/
and a nice animation
http://www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.html

The other source of information are research articles and reviews publish in scientific journals. Pubmed is the search engine http://www.ncbi.nlm.nih.gov/entrez/query.fcgi . I, however, doubt that you will have access to most of the journal and that you will understand what the authors are talking about.

If you don't understand some concept, do not be shy, ask questions.

2.) I’m familiar with the petunia that was put through RNAi and eventually turned white rather than a deep purple when gene copies of key enzymes for flower pigmentation were introduced. What I don’t understand is how I could replicate this in the lab or something possibly simpler? If someone has a source of the procedures could someone inform about how to get them?

There is several possible application for RNAi. It range from using it a tool in experiement for biologist to using it to cure cancer and some infections.

It is quite easy to replicate in lab in simpler organism like bacteria. I am doing some sort of RNAi work. Some my procedure is simple. I found a gene/protein that is required by the bacteria and have the complete genetic sequence of that gene. So I amplify the gene by means of PCR (http://www.people.virginia.edu/~rjh9u/pcranim.html[/URL] ; [PLAIN]http://faculty.plattsburgh.edu/donald.slish/PCRmov.html[/URL]) and I insert the product upstream of an inducible promoter on a plasmid ([url]http://www.bio.davidson.edu/courses/genomics/method/plasmid_inducible.html[/URL]). The trick is to insert the gene in the reverse direction relative to the promoter (if you look at the figure from the last link, the green arrow would be pointing the other way). Therefore when gene expression is induce, there will be an antisense mRNA (ASmRNA) that will bind with the normal sense mRNA and interfere with the translation and with the binding of the ribosme onto the mRNA.

So when I do my experiment, I take two bacterial culture, one in which the ASmRNA is not induce and the other has the ASmRNA induce and measure the growth. Because I am studying a require gene, there will be no growth in the culture with an induce ASmRNA and plenty of growth in the non-induce culture. The ASmRNA is stopping/slowing the growth of the bacteria

For your project, you may not want to use a required gene. Some bacteria produce pigment and the pigmentation could be knock out.

 

[DocToxyn]

I don't have any direct experience with RNAi, siRNA, etc, but perhaps I can help out. First, how long do you have to complete this project and what resources do you have available? This may be the critical determining factor as to whether you can pull this off. Having said that, I'm sure you can find some review and/or method articles out there that can get you started, try a search for "RNAi method" in PubMed, I found this and this. That should get you started.

Are you near any universities? This may be a great resource for you. If you can find a researcher who has done this and can at the very least give you some direction, if not invite you into his/her lab, that would take you a long way. Along the way you may find some other aspect of genetics that appeals to you more or is more suitable for your specific timeframe/resources. Good luck and don't hesitate to come back with more questions.

 

[Garret]

Usually the best start is to find a intro. to genetics book for university. However, RNAi has only been popular in the last few years, so it might not be included in the book.

It just happens to be that I have the upcoming Friday off and well my friend invited me to attend some of his classes at the university so while I’m there I’ll take out the textbook, “RNA interference technology: from basic science to drug development,” and “RNAi: a guide to gene silencing.” It’s not too uncommon for me to take out books from the local campus. I’ll probably have some difficulty understanding everything but I’ll be sure to ask questions.

There is several possible application for RNAi. It range from using it a tool in experiement for biologist to using it to cure cancer and some infections.

It is quite easy to replicate in lab in simpler organism like bacteria. I am doing some sort of RNAi work. Some my procedure is simple. I found a gene/protein that is required by the bacteria and have the complete genetic sequence of that gene. So I amplify the gene by means of PCR (http://www.people.virginia.edu/~rjh9u/pcranim.html ; http://faculty.plattsburgh.edu/donald.slish/PCRmov.html ) and I insert the product upstream of an inducible promoter on a plasmid (http://www.bio.davidson.edu/courses/genomics/method/plasmid_inducible.html). The trick is to insert the gene in the reverse direction relative to the promoter (if you look at the figure from the last link, the green arrow would be pointing the other way). Therefore when gene expression is induce, there will be an antisense mRNA (ASmRNA) that will bind with the normal sense mRNA and interfere with the translation and with the binding of the ribosme onto the mRNA.

So when I do my experiment, I take two bacterial culture, one in which the ASmRNA is not induce and the other has the ASmRNA induce and measure the growth. Because I am studying a require gene, there will be no growth in the culture with an induce ASmRNA and plenty of growth in the non-induce culture. The ASmRNA is stopping/slowing the growth of the bacteria

For your project, you may not want to use a required gene. Some bacteria produce pigment and the pigmentation could be knock out.

Well I’m unsure what I would be doing in the lab; I need to think of something novel. As far as what you’ve explained I get most of it. I’m unsure how I would isolate a gene sequence to put it through PCR? And I’m also a little unsure on what it would take in terms of lab instruments to insert the product upstream?

Overall I’m excited to learn more and actually try this out in the lab. Also, thanks for your help; you are truly the Uber Mentor.

Regards,
Garret

 

[Garret]

I don't have any direct experience with RNAi, siRNA, etc, but perhaps I can help out. First, how long do you have to complete this project and what resources do you have available? This may be the critical determining factor as to whether you can pull this off. Having said that, I'm sure you can find some review and/or method articles out there that can get you started, try a search for "RNAi method" in PubMed, I found this and this. That should get you started.

Are you near any universities? This may be a great resource for you. If you can find a researcher who has done this and can at the very least give you some direction, if not invite you into his/her lab, that would take you a long way. Along the way you may find some other aspect of genetics that appeals to you more or is more suitable for your specific timeframe/resources. Good luck and don't hesitate to come back with more questions.

I have about 6 months. I looked at your links and will definitely use them as a catalyst to my research, thanks. I’ll see if I can find a professor to show me some of the basics. I have dealt with a professor at the university once before with the extraction of Lycopene. I’ll probably ask the head of the science department for some help finding contacts, I know him quite well so he will definitely help me out. Your definitely right about how I’ll probably find something else/similar that will appeal, it happens a lot! Thanks for the advice.

Regards,
Garret

 

[iansmith]

Well I’m unsure what I would be doing in the lab; I need to think of something novel. As far as what you’ve explained I get most of it. I’m unsure how I would isolate a gene sequence to put it through PCR? And I’m also a little unsure on what it would take in terms of lab instruments to insert the product upstream?

For a project like yours, you will need to get access to a university lab that do molecular biology.

You do not need to isolate a gene. You need to isolate genomics/chromosomal DNA from the organism of interest. For the sequence of the gene, if the genome of the organism has already been fully or partly sequenced, the information is available in databank on the web. The database is called genbank on the website is http://www.ncbi.nlm.nih.gov/

The next step is create the primer/oligonucleotides that will amplify the desired section. To do PCR, you need special equipement that is usually available in labs that do molecular biology at a university.

To introduce to gene in a plasmid, you do not need special equipment but you need specific enzymes and solutions. One set of enzyme, called restriction enzyme, will create sticky ends that will allow both DNA fragment to stick together (ttp://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/recombinant/enuc.html). The other enzyme, called ligase, will ligate both DNA fragment. Most of the enzyme require for this procedure are available at a lab that do molecular biology.

 

[Genecks]

Don't know if you tried these..
Google Define
Wiki
http://findarticles.com/p/search?qt=RNAi&tb=art&qf=health

 

[Moonbear]

With a 6 month time frame, you'll probably want to find out about doing a sub-project of something already in progress in a university lab. You won't have enough time to really trouble-shoot and develop new RNAi for something totally novel, but you will have time to do a smaller scale project if someone has already gotten RNAi developed for a particular gene target and is now going to test the function of the gene by suppressing it.

 

[sssddd]

woah woah back up a second. did you even get a lab for yourself in the first place?
And does this lab have all the reagents necessary for this kind of experiment. It would be bad if you have to buy all these expensive equipments and these expensive enzymes by yourself.

 

[Moonbear]

woah woah back up a second. did you even get a lab for yourself in the first place?
And does this lab have all the reagents necessary for this kind of experiment. It would be bad if you have to buy all these expensive equipments and these expensive enzymes by yourself.

Yep, that's why we're all pretty much agreed about telling Garrett he needs to find someone at a university to work with for this project (and why I've suggested it should be a sub-project of ongoing research). It's not just challenging but expensive to work with these types of methods.