Using SCGE to Determine the Effectiveness of Chemoprevention from SDG

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KurtisSWC
Posts: 3
Joined: Sat Dec 03, 2016 3:50 pm
Occupation: Student
Project Question: Using Single Cell Electrophoresis to Determine the Effectiveness of Chemoprevention from SDG
Project Due Date: April 2017
Project Status: I am conducting my research

Using SCGE to Determine the Effectiveness of Chemoprevention from SDG

Post by KurtisSWC »

Hello there,

My name is Kurtis, I am a level II student, This year, I am doing a paper on 'Using Single-Cell Gel Electrophoresis to Determine the Effectiveness of Chemoprevention from SDG". SDG (secoisolariciresinol diglucoside) is a lignan found in flax seed, which has been studied for its potential anti-carcinogenetic properties.

The objective of my project is to use ‘single-cell gel electrophoresis” (Comet Assay), to analyse the extent of DNA damage found in of both a basal yeast culture (control) and of a basal yeast culture administered secoisolariciresinol diglucoside (SDG), once exposed to a certain carcinogen.

I have conducted some background research into the properties of SDG, yet I have only limited access to journals. As of this moment I have recorded how I will culture the yeast, and analyze the cells via SCGE. But I do not have the slightest clue on how to administer SDG to one culture of yeast, wherein it will produce some effect, yet not kill the cells due to change in pH (as that is what I have encountered during research). I also do not have any background information on a specific mutagen to use to inflict DNA damage, as a specific mutagen may vary on the interest of my mentor. (never found one yet, I am searching at my local university).

If anyone has some great sources or input for what method to introduce SDG to my culture, it would be greatly appreciated! I was hoping to figure this out last week, but being in advanced courses I haven't the time for much extracurricular research. My teachers were mentioning applying for a competition called the Sanofi BioGenius Competition, but the deadline is tomorrow. I need a detailed description of the experimental process to apply- something incomplete if I dont know how to administer SDG (or if it would even yield results).

I am still going to do this experiment for my school science fair and the regional competition, so if anyone has any input that would be amazing.
Last edited by KurtisSWC on Sun Dec 04, 2016 1:10 pm, edited 1 time in total.
donnahardy2
Former Expert
Posts: 2671
Joined: Mon Nov 14, 2005 12:45 pm

Re: Using SCGE to Determine the Effectiveness of Chemoprevention from SDG

Post by donnahardy2 »

Hi Kurtis,

Will you please edit your post to delete your last name? You should not include any personal information on this website that would allow anyone to find you. This is a safety rule designed to protect you. We definitely want to help with your science project, but communication has to be anonymous.

Your science project idea is really excellent. This is a unique idea and your questions are good and this will help in winning at the science fair. Since the deadline for entering the competition is today, then use the protocol from the following project as an outline for your experiment. This experiment will allow you to measure the antifungal properties of SDG. You will need to edit the method to make it specific for your experiment.

https://www.sciencebuddies.org/science-f ... p012.shtml

Do you have any references for your topic? I think you realize that you do need some references that will support your project idea. I will look and see if I can find anything and post again as soon as possible.
Let me know if you have any questions.

Donna
donnahardy2
Former Expert
Posts: 2671
Joined: Mon Nov 14, 2005 12:45 pm

Re: Using SCGE to Determine the Effectiveness of Chemoprevention from SDG

Post by donnahardy2 »

Hi Kurtis,

I am having the same problem that you had in finding research articles that I can access. However, you may be able to use the following for your bibliography:

http://pubs.acs.org/doi/abs/10.1021/jf0005871

This paper reports an HPLC method for Secoisolariciresinol diglucoside analysis. Looking at the structure of the molecule, I think the method may use a reverse phase HPLC column. If you have access to an HPLC system, I could do more searching on this possibility.

The Wikipedia article on SDG has several articles with access that contain information that should be useful:

https://en.wikipedia.org/wiki/Secoisola ... iglucoside

Here is a good article that is a review of other published papers on SDG:
https://nutritionj.biomedcentral.com/ar ... 015-0059-3

For your project proposal, be sure to include the background information from this article that includes reasons for investigation SDG. A good scientific reason along with a valid experimental protocol should help get your project idea accepted. You will then need to work hard to get the project completed.

I hope this helps. Please post again if you have any other questions.

Donna Hardy
KurtisSWC
Posts: 3
Joined: Sat Dec 03, 2016 3:50 pm
Occupation: Student
Project Question: Using Single Cell Electrophoresis to Determine the Effectiveness of Chemoprevention from SDG
Project Due Date: April 2017
Project Status: I am conducting my research

Re: Using SCGE to Determine the Effectiveness of Chemoprevention from SDG

Post by KurtisSWC »

Hello,

I have now accumulated many articles on SDG. Would I be allowed to upload the cited research I have conducted?

If not, here is the breakdown of what I have found.

-SDG inhibits the process of carcinogenesis, as well as reducing size of tumors.
- how to complete the Comet assay.
-how to culture cells.
-how to analyze the results of single cell electrophoresis.

Now I said I was going to use yeast, but I have done some research and I have seen an article state that yeast will not produce desired results in single cell electrophoresis, as they contain a cell wall (single cell electrophoresis requires cells to undergo lysis to let DNA unravel and create 'tails'). Now I am almost back to square one. I have decided to turn down the biogenius competition, and just go to the regional science fair in my province.

I have contacted a doctor who wrote an article about SDG, and I'm awaiting their advice for what cell type to culture, and how I could possibly administer the compound SDG to it.

Essentially, those two questions I need answered to go ahead with the rest of the project.

As of right now I am creating a template for my journal, and filling it with my background research, template abstract, etc. I have contacted a few possible mentors and now I am just in the waiting process. I will update as soon as I get any information.


Thanks,

Kurtis
donnahardy2
Former Expert
Posts: 2671
Joined: Mon Nov 14, 2005 12:45 pm

Re: Using SCGE to Determine the Effectiveness of Chemoprevention from SDG

Post by donnahardy2 »

Hi Kurtis,

Thanks for your reply and for deleting your last name. I realized after I posted my last comments that I had not commented on the Comet assay. I did not understand how the comet assay would fit into your plan.

It's really great that you have done background research. Please do post the information that you have found.. If you include the internet links, I should be able to open the references and understand what you want to do. You can also upload attachments on this forum, but it will take a little longer.

Please let me know what your objective is. Have you defined your project question? I can probably help recommend an experimental protocol that will make sense. It sounds like you just need to find a suitable cell for the experiment and define a few other details.

It would be great if you could get a local mentor who is a subject expert on SDG, but you will still be able to do a great project if this is not possible.

Let me know if you have access to a lab and the electrophoresis equipment you will need for the comet assay.

Donna
KurtisSWC
Posts: 3
Joined: Sat Dec 03, 2016 3:50 pm
Occupation: Student
Project Question: Using Single Cell Electrophoresis to Determine the Effectiveness of Chemoprevention from SDG
Project Due Date: April 2017
Project Status: I am conducting my research

Re: Using SCGE to Determine the Effectiveness of Chemoprevention from SDG

Post by KurtisSWC »

Hello,

I am going to put up all of the background info I put together, so you may get a more accurate idea on this project. Some research was done in books and physical documents, so you may have to search them.


Background Information
---
Properties of SDG:


A free radical is a species containing one or more unpaired electrons[1]. Free radical reactions are reactions that occur as a part of homeostasis, where an electron detaches from a molecule and then reattaches almost instantaneously[2]. Because of this, free radicals can become harmful to the human body if they do not attach to an antioxidant. An antioxidant is a substance such as vitamin C, that removes potentially damaging oxidizing agents in a living organism. Unsatisfied free radicals can spur the mutation of cells they encounter and are, thus, causes of cancer. Antioxidants, being able to remove these free radicals ultimately possess the ability to prevent cancer. “The main flax lignan secoisolariciresinol diglucoside (SDG) is an antioxidant. It scavenges for certain free radicals like the hydroxyl ion (•OH). Our bodies produce free radicals continually as we use (oxidize) fats, proteins, alcohol and some carbohydrates for energy. Free radicals can damage tissues and have been implicated in the pathology of many diseases like atherosclerosis, cancer and Alzheimer disease[3].” “(An) anticarcinogenic effect of SDG molecule has been observed in pulmonary metastasis, mammary gland and breast cancer metastasis. Studies have shown that the supplementation of SDG in mice diet resulted in reduction of volume, area and numbers of tumors significantly as compared a control mice group [4].” “In a study just published in BMC Cancer, researchers found that a diet of flaxseed given to mice not only protects lung tissues before exposure to radiation, but can also significantly reduce damage after exposure occurs [5].” Ultimately, SDG has shown its anti-carcinogenic properties, and a promising area of study for early cancer prevention/reduction.



Background Information
---
Single Cell Electrophoresis:

“The Comet Assay, also called single cell gel electrophoresis (SCGE), is a sensitive and rapid technique for quantifying and analyzing DNA damage in individual cells. As such, this is one of the techniques used in the area of cancer research for the evaluation of genotoxicity and effectiveness of chemoprevention. Swedish researchers Östling & Johansson developed this technique in 1984.1 Singh, et al., later modified this technique, in 1988, as the Alkaline Comet Assay.2 The resulting image that is obtained resembles a "comet" with a distinct head and tail. The head is composed of intact DNA, while the tail consists of damaged (single-strand or double-strand breaks) or broken pieces of DNA [6].” “Compared to other genotoxicity assays, the advantages of this technique include: (1) high sensitivity for detecting low levels of DNA damage; (2) ability to detect genotoxicity in the absence of mitotic activity; (3) the requirement for small numbers of cells per sample; (4) flexibility; (5) low costs; (6) easy application; and (7) the relatively short time period (a few days) needed to complete an experiment [7].”

The Process:

“Individual cells are embedded in a thin agarose gel on a microscope slide. All cellular proteins are then removed from the cells by lysing. The DNA is allowed to unwind under alkaline/neutral conditions. Following the unwinding, the DNA undergoes electrophoresis, allowing the broken DNA fragments or damaged DNA to migrate away from the nucleus. After staining with a DNA-specific fluorescent dye such as ethidium bromide or propidium iodide, the gel is read for amount of fluorescence in head and tail and length of tail. The extent of DNA liberated from the head of the comet is directly proportional to the amount of DNA damage [6].”



Objective:
---

To use the biotechnological technique ‘single-cell gel electrophoresis” (Comet Assay), to analyse the extent of DNA damage found in of both a basal yeast culture (control) and of a basal yeast culture administered secoisolariciresinol diglucoside (SDG), once exposed to (level) of (mutagen).



Relevant Application:
---

It's estimated that in 2016, 25,700 women were diagnosed with breast cancer (“Canadian Cancer Society”, 2016). The compound secoisolariciresinol diglucoside (SDG), has been observed in studies showing that a supplementation of SDG in mice diets resulted in the reduction of volume, area and numbers of metastatic breast cancer tumours significantly as compared to mice without SDG (Li, Yee, Thompson, & Yan, 1999). Ultimately, the compound SDG has shown an anti-carcinogenic quality, which can be further tested to create antioxidant drugs or techniques for preventing cancer. This project is an in vitro assessment of the qualities SDG holds, by analysing its ability to prevent DNA damage in individual cells. Using data obtained from individual cells, we can observe the direct role SDG serves in inhibition of carcinogenesis. If the results turn out to be what was hypothesised, further tests on this compound can be done to determine exactly how SDG prevents the process of carcinogenesis; potentially paving a new path in early cancer prevention, without the use of genotoxic treatments such as radiation therapy. SDG could potentially reduce the number of secondary malignancy cases, due to such treatments.



Hypothesis
---

I hypothesize that once electrophoresed, the cells collected from the yeast culture administered the compound SDG, will have smaller ‘tails’ in the electrophoresis chamber. The cells from the control yeast culture will have larger ‘tails’. These results would indicate that SDG had a role in preventing the process of carcinogenesis, demonstrating SDGs chemopreventive quality.



Bibliography
---

1. N.d. "CHE 230 Homepage." Retrieved December 4, 2016 (http://www.chem.uky.edu/courses/che230/fl/).

2. Cashin-Garbutt. 2013. "What are Antioxidants?" News-Medical. Retrieved December 4, 2016 (http://www.news-medical.net/health/What ... dants.aspx).

3. 2015. "Research." Flax Council Of Canada. Retrieved December 4, 2016 (http://flaxcouncil.ca/resources/research/).

4. Li D, Yee JA, Thompson LU. Dietary supplementation with secoisolariciresinol diglycoside (SDG) reduces experimental metastasis of melanoma cells in mice. Cancer Lett. 1999;142:91–6.

5. Christofidou-Solomidou, M., Tyagi, S., Tan, K.-S., Hagan, S., Pietrofesa, R., Dukes, F., … Cengel, K. A. (2011). Dietary flaxseed administered post thoracic radiation treatment improves survival and mitigates radiation-induced pneumonopathy in mice. BMC Cancer, 11(1), . doi:10.1186/1471-2407-11-269

6. Privacy. (2016). Comet assay | single cell gel electrophoresis. Retrieved December 1, 2016, from http://www.sigmaaldrich.com/life-scienc ... assay.html

7. What is better experimental design for in vitro comet .. (2008). Retrieved December 1, 2016, from http://fliphtml5.com/glpa/akhn/basic





My tasks are to find:

a mutagen to use.
new cell type able to be electrophoresed.
how to administer SDG to cultures.

As you can see from my research on antioxidants, they prevent free radicals from damaging DNA. My chemistry teacher let me borrow her thesis to study how she cultured her cells In her study. I have seen she had oxidized a porous silicon using hydrogen peroxide to use as the culture media, as that was her topic- that raised a question in me. Can I use hydrogen peroxide to inflict the DNA damaged on my test cells- that way the SDG can work on removing the free radicals they produce? I will do some more research into if this is the best oxidant to use. It seems practical, as it is cheaper- every doctor will have this available for use when I conduct my experiment too.

I also asked my chemistry teacher how should I search for a cell type to use, and she told me to wait until I have secured a mentor as they will likely choose the cell type based on what they have in their labs. So that is taken care of.

Now the only problem I have as a barrier to start the actual preparation of the experiment is how I will let the cells take the SDG. I seen another paper which the scientists used SDG as part of their culture media, so I will email them this question along with my project outline tonight and hopefully they get back.
donnahardy2
Former Expert
Posts: 2671
Joined: Mon Nov 14, 2005 12:45 pm

Re: Using SCGE to Determine the Effectiveness of Chemoprevention from SDG

Post by donnahardy2 »

Hi,

Thanks for posting the details of your project proposal; this was very helpful for me to understand what you are doing.
Your background information and project proposal to use the comet assay are impressive and using the comet assay will allow you to measure the results. Your questions are good’ it’s great that you are working on the final details of the experimental design at this point.

To add the SDG to your sample, you definitely want to add it to the tissue culture medium when you initially transfer the cells to new medium. You will need to do different concentrations, at least 3 plus a control with no SDG, if possible. In the research article you found, did the authors mention the concentration of SDG used? If so, then use a similar concentration. It’s important to test different concentrations so you can see a dose effect.

Do you have SDG, or are you going to use flax seed oil? If you are using flax seed oil, do you have a way to measure the SDG in the sample so you will know exactly how much you will be adding?

Hydrogen peroxide is a good oxidizing agent and is definitely as possibility. You can search for reference articles that include assays for antioxidants to find out about other possibilities.

Here is a reference that used a photosensitizer Ro19-8022 and light to induce oxidation. (refer to Figure 1). Also, look at Figure 2, which shows results after adding hydrogen peroxide and using the comet assay to measure results. I haven’t had a chance to read this complete article yet, so I’ll look for more information.

http://ajcn.nutrition.org/content/81/1/261S.full

I’ll look for more references, and you should do the same. Ir's best to find out what other researchers have done in the past, and then start your project from that point.

I hope that the researcher using SDG will respond to your inquiry, as this would provide you with the best information, and I hope you can find a lab to do the experiment in also. This is an exciting project, but there is lots of lab work to be done.

Donna
KSWC179
Posts: 1
Joined: Wed Feb 22, 2017 7:31 am
Occupation: Student

Re: Using SCGE to Determine the Effectiveness of Chemoprevention from SDG

Post by KSWC179 »

Hello there,

I'm Kurtis, but I couldn't log in to my old account. I would like to update you on the status of the project, since we last spoke.

Apon meeting my mentor, he had assigned a graduate student to work alongside me in my project, and we have discussed a change in the layout of the project. We have chosen to use the qPCR assay to measure the difference between DNA damage, rather than SCGE. we have also chose to use the human CCD-18Co (colon) cell line, as that is where SDG gets absorbed. Our oxidizer is still hydrogen peroxide. And more good news; my application for the Sanofi Biotechnology Competition was accepted so I am officially going.

The way the project will be completed is as follows:

We will culture the cells and transfer equal amounts to microcenterfuge tubes. Then we will inoculate SDG at parameters of 0, 10, 50, and 100 mM (micromolar). The cells will be treated for 24 hours (constant), then be introduced to 1mM of hydrogen peroxide, for 15 minutes (constant). We will complete the experiment again with dosages of hydrogen peroxide of 3 and 5 mM, to visualize a pattern in protection. Once the 15 minutes are up, the cells will get rinsed, and then we will extract the DNA of each parameter via a DNA extraction kit in the lab. We then will run each parameter in PCR. PCR is a biotechnological technique that quantifies DNA over several orders of magnitude. That being said, any DNA leisons or damage will hault the process of PCR, and lessen the amount of quantified DNA. we can notice the difference of healthy and damaged DNA as the more result DNA present, the less damage- vice versa.

Now is where the aspect of my previous project comes into play. To visualize the results in a feasible manner, we will stain the DNA parameters with the bio fluorescent dye SYBR Safe, and then run each parameter in standard gel electrophoresis. The way results will be recorded is that (amount of dye = amount of DNA = amount of DNA damage).

The experiment will be conducted multiple times and then results averaged out. If you have any questions of input for this assay, please feel free to reply. I appreciate any advice.


Sincerely,

Kurtis
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