Chapter 7: BIOL 302L Research Projects

An important consideration of our research project is to keep in mind that the goal of any research should be to add to the scientific body of knowledge. Ask the question, what contributions may be made by addressing an area or areas that are not known, not limited by the experimental time constraints for this laboratory. For instance, we know that the HL-60 cells may be induced to differentiate into different cell types with different functions in the body. If, by understanding what the mechanisms that control this differentiation are, we may ultimately have a better understanding of how a disease may be controlled or eliminated. Recent publications in diverse areas of research involving MMP-9 and disease include cancer, neurological, gastric, cardiac, pharmaceutical development, and dental investigations. We strongly encourage all to investigate extensively these areas of research before making the decision as to which research project to pursue.

To begin our research project, we have been asked to find a primary journal article or articles that 1) bases its research using the HL-60 cell line or may be modified to use this cell line and, 2) employs methodology that is available in this laboratory (list provided for us below). There are two important aspects to consider when designing a research project 1) the model organism to use and, 2) scale of the experiment that will address and answer the research question asked. For this laboratory we will only be using the HL-60 cell line for our model organism and our experimental design may be selected from the available procedures or methods.

We will then consider what could be an appropriate question (our working scientific hypothesis) for us to ask for our research project. Our graduate instructional assistant will assist us and give us approval for following this line of questioning for our research. Once we know the question we want to ask, we need to determine the experimental method that will give us results that will answer that question.

Consider:

1. What physical or physiological components measured would be appropriate to examine that might yield those results?

2. Are the protocols established in the literature? Which one can we select to perform our experiment and collect our data? Is it directly relevant to our research question?

3. If the experimental approach is not established in the literature, can we use a procedure that deviates minimally from established procedure? Remember, our experimental approach must give us valid results.

4. Does our experiment require living cells (in vivo sort of lol) or cellular components (in vitro) in order to obtain the desired results?

Comparing in vivo and in vitro experimental approaches:

In vivo: uses living organisms or cells; tests whole or multiple different interacting cell types, or depends upon the organism or cellular interaction with the environment.

In vitro: involves precise control of biological product levels and the interaction mechanism can be fully replicated; requires disassembling the living cell into constituent components and understanding how many cell components are needed to answer the research question.

Link the research project to previous research. Are the methods used in this paper employing in vivo or in vitro approaches? Immunoblotting and Gelatin Zymography, Stable Transfection of Cells, RNA Isolation and Northern Analysis. 

Instrumentation Selection

Level of scale necessary to build an experimental protocol to answer fundamental research questions. How to choose instrumentation to answer questions. The technology available in this laboratory allows us to examine cells microscopically or using analytical instrumentation (spectrophotometer, fluorometer, or electrophoretic analysis). We will need to decide if the research question we are asking can be answered and give valid results using a specific procedure or method. This selection must be determined based on addressing the research question, not on the methodology available.

1. Microscopy vs. analytical instrumentation, i.e. spectrophotometer, fluorometer, or electrophoretic analysis.

2. Microscope: analysis may be performed using light or fluorescent methodology.

3. Fixed tissue and any dye (in vitro) vs living cell and vital fluorescent dye (in vivo).

Separation of cellular components to examine changes: organelles or molecular level? 

1. Ask a question.

A. Search the literature.

B. Determine what is known or unknown about the topic of interest.

2. Formulate a hypothesis.

A. Make a prediction based on research

B. Include both null and alternative hypotheses.

C.   Determine the available methods and reagents.

3. Design an experiment.

A. Determine if other methods and reagents are feasible.

B. Design experimental plan – think about controls and replication.

4. Data collection.

A. Decide on methodology.

B. Determine controls and replication.

C.    Determine method of data analysis.

5. Write a proposal.

A. Background

B. Experimental design.

C.    Materials needed – needs instructor approval.

6. Present proposal (Powerpoint).

A. Background

B. Hypothesis

C.    Methods

7. Carry out the experiment.

A. Need detailed notes on materials and methods, results, and observations.

B. Include controls (positive and negative) and replication.

8. Write a scientific poster to report data.

A. Use rubric as a guide ( more information on blackboard, how to present a poster).

B. Include relevant graphs, charts, pictures, or formulas.

Begin primary literature search.

• See reagent list (listed below, and as separate document on Blackboard) and protocol lists (above) to confirm availability of protocol and reagents.

• If reagent is not on the list, prepare a written request to give to your GIA to see if it can be ordered. If this is a remote lab, assume we cannot order it in time and please select some of the listed reagents present.

Presentation of research projects to GIA.

• We will have a conversation with our GIA to fill out a form that outlines our exact research project. This will help ensure all group members know what we're up to and the GIA has plenty of time to relay cell work needs to the Coordinator. 

• Be sure to explain to our GIA what makes our project different from the other experiments we have done in the class. What is the different reagent we are using and/or if the protocol we are using has not already been performed by the class, explain the protocol and what it shows.

• Please see Blackboard for a more in depth rubric.

Proposal for research projects due.

• We'll craft our research proposals after the Magellan Scholar proposal! It's a short proposal that covers all the important parts: Background (Introduction) which should cover the information for the reviewer to be up to date on information in the field, Research Question, Project Goal and Objectives, Project Impact, Significance, or purpose, Project Design and Methods, Project Timeline, Anticipated Results, a group personal statement, and References section. For more information, check out the OUR website: https://sc.edu/about/offices_and_divisions/undergraduate_research/apply_for_funding/our_funding/magellan-scholar-award/applying/index.php

Complete the research project

• Working collaboratively, complete your research project, collecting and analyzing the data from your project. This may take a few class periods and may entail your whole group coming to lab or it may be completed quickly in one period. If we have any issues with our group, please contact our GIA quickly!

Finishing your project

• After collecting the data, it’s time to analyze it, draw our conclusions, and write it up! Remember the final research poster will be a group effort. Don’t forget to follow the rubric on blackboard to ensure an amazing grade! 

Finally, make sure to bask in all that is science! Go us, go science!

Here’s a small review of the more detailed protocols that follow below. Feel free to use more than one.

Apoptosis:

• Apoptosis detection using caspase kit (fluorometric glo 3/7)

• Apoptosis detection using DNA isolation and DNA fragmentation protocol

Gene Expression:

• mRNA analysis using RT-PCR

Protein Activity:

• Enzyme analysis using zymography gel electrophoresis

Morphology Analysis:

• Differentiation of HL-60 cells using May Grunwald Stain