20.109(S20):Prepare cells for RNA purification and practice RNA-seq data analysis methods (Day1)

Introduction

We will use the DLD-1 cell line in this module to examine gene expression in cancer cells. The DLD-1 cell line originated from the colon of a male patient with colorectal adenocarcinoma Dukes Type C. In the figure to the right, it is shown that cell survival decreased as higher concentrations of a DNA damaging agent, etoposide, were added.

Image modified from Hucl et al. PMID:18593900

You will work with these cells, mostly, in our tissue culture room. Tissue culture was developed ~100 years ago as a means to study mammalian biology, and since that time we have learned a tremendous amount by observing the behavior of mammalian cells maintained in the laboratory. The term tissue culture was originally coined because researchers were doing exactly that, extracting tissue and letting it live in a dish for a short time. Today, most tissue culture experiments are done using cells rather than tissues. Much of what we know about cancer, heritable diseases, and the effects of the environment on human health has been derived from studies of cultured cells.

What types of cells do people study, and from where do they come? Cells acquired directly from animal tissue are called primary cells. They are difficult to culture, largely because primary cells in this context divide only a limited number of times. This limitation on the lifespan of cultured primary cells, called the Hayflick limit, is a problem because it requires a researcher to repeatedly remove tissues from animals to complete a study. Cell isolation processes can be quite labor-intensive, and also can complicate data analysis due to inherent animal-to-animal variation. To get around this problem, researchers study cells that are immortal, which means the cells are able to divide indefinitely. Though using immortal cells is preferable for many reasons, some inherent cell-to-cell variation still exists in such populations and the genetic changes that cause immortality may affect experimental outcomes.

The art of tissue culture lies in the ability to create conditions that are similar to what a cell would experience in an animal, namely 37°C and neutral pH. Blood nourishes the cells in an animal, and blood components are therefore used to feed cells in culture. Serum, the cell-free (and clotting-factor free) component of blood, contains many of the factors necessary to support the growth of cells outside the animal. Consequently, serum is frequently added to tissue culture medium, although serum-free media (also called chemically defined media) exist and support some types of cultured cells.

Cultured mammalian cells must grow in a germ-free environment and researchers using tissue culture must be skilled in sterile or aseptic technique. Bacteria double very quickly relative to mammalian cells. An average mammalian cell doubles about once per day whereas many common bacteria can double every 20 minutes under optimal conditions. Consequently, if you were to co-culture 100 mammalian cells and 1 bacteria together in a flask, within 24 hours you would have ~200 unhappy mammalian cells, and about 100 million happy bacteria! Needless to say, you would not find it very useful to continue to study the behavior of your mammalian cells under these conditions.

Protocols

Part 1: Practice tissue culture techniques

As you prepare to work in the tissue culture room, review the following resource:

• General dos and do nots of working in the tissue culture laboratory

Part 1a: Demonstrate best practices

The teaching faculty will show you several key techniques that will be important to your success in tissue culture. Please carefully observe the following procedures, take notes, and ask questions!

1. Prepare the tissue culture hood.
2. Observe your cells.
3. Trypsinization of cultured cells.
4. Calculate cell culture density.
5. Seed cells.
6. Clean tissue culture hood.

Part 1b: Seed cells for RNA purification

You will use the techniques you learned during the demonstration to seed T75 flasks with cells. These cells will be used to assess the effect of etoposide on gene expression in DLD-1 cells.

Each team will receive one T25 culture flasks with DLD-1 cells. You will enzymatically detach these adherent cells, count them, and seed them at specific densities into fresh T25 flasks to prevent the cells from overgrowing.

Preparing tissue culture hood

1. The tissue culture hood is partly set up for you. Finish preparing your hood according to the demonstration, first bringing in any remaining supplies you will need, then obtaining the pre-warmed reagents from the water bath, and finally retrieving your cells from the 37 °C incubator.
   • Don't forget to spray everything down with 70% ethanol!
   • One of the greatest sources for tissue culture contamination is moving materials in and out of the hood because this disturbs the air flow that maintains a sterile environment inside the hood. Think about what you will need during your experiment to avoid moving your arms in and out of the hood while you are handling your cells.

Collecting cells

1. Examine your cell cultures as you remove the flasks from the incubator.
   • Look first at the color and clarity of the media. Fresh media is reddish-orange in color and if the media in your flask is yellow or cloudy, it could mean that the cells are overgrown, contaminated, or starved for CO₂.
   • Next, look at the cells using the inverted microscope. Note their shape, arrangement, and how densely the cells cover the surface of the flask.
2. After you look at your cells, take the flask to your tissue culture hood to begin the seeding procedure.
3. Aspirate the media from the cells using a sterile Pasteur pipet.
4. Wash the cells by adding 2 mL PBS using a 5 mL pipet. Slightly tip the flask back and forth to rinse the cells then aspirate the PBS with a fresh Pasteur pipet.
5. To dislodge the cells from the flask, you will add trypsin, a proteolytic enzyme.
   • With a 2 mL pipet, add 1 mL of trypsin to the flask.
   • 2 mL pipets are tricky! They fill up quickly. Be careful not to pull up the liquid too quickly or it will go all the way up your pipet into the pipet-aid! If this happens, please alert the teaching faculty rather than returning the pipet-aid to the rack.
6. Tip the flask in each direction to distribute the trypsin evenly then incubate the cells at 37°C for 3 minutes using a timer.
   • This is a great time to clear out your trash and read ahead!
7. Retrieve your flasks from the incubator and firmly tap the bottom 10 times to dislodge the cells.
   • Check your cells using the microscope to ensure they are dislodged. They should appear round and move freely.
   • If your cells are not detached from the flask, incubate at 37 °C for an additional minute.
8. When your cells are dislodged, move your flask back into the tissue culture hood and add 3 mL of media to the cells then pipet the liquid up and down (“triturate”) to break up cells that are clumped together and suspend them in the liquid.
   • Note: do not take up or release all the liquid, in order to avoid bubbles.
9. Transfer the suspended cells into a labeled 15 mL conical tube
10. Transfer 90 μL of your cell suspension from the 15 mL conical tube into a labeled eppendorf tube.
    • Be sure to cap your conical tube and eppendorf tube after you transfer your cells.

Counting cells

1. Remove the eppendorf with your cells from the tissue culture hood.
2. Add 10 μL of Trypan blue to the eppendorf tube and pipet up and down to mix.
3. With a new pipet tip, carefully pipet 10 μL of the stained cells between the hemocytometer and (weighted) glass cover slip, as shown to you by the teaching faculty.
4. Count the cells that fall within the four corner squares (with a 4x4 etched grid pattern), average (i.e. divide by 4), and then multiply by 10,000 to determine the number of cells/mL.

Seeding cells

1. Obtain one T25 flasks and label DLD-1.
   • Include your section information and the date.
2. Calculate the volume of the cell suspension you need to seed 250,000 DLD-1 cells in the T25 flask.
   • For example, if your concentration is 1 million (1 M) cells/mL, you would seed one fresh T25 flasks with 250 μL of DLD-1 cells.
3. Subtract the volume of cell suspension that you calculated in Step #2 from 5 mL.
   • Add this volume of media to the fresh T25 flask.
4. Add the volume of cell suspension that you calculated in Step #2.
   • You should mix your cell suspension by pipetting before distributing the small volume of cells to the flasks as the cells likely settled to the bottom of the conical while you were counting.
5. Finally, tilt your flasks back and forth to distribute the cells evenly.
6. Before moving your flasks to the 37 °C incubator, use the microscope to visually confirm that cells are present.

The teaching faculty will use your cultures to seed larger flasks, T75 flasks, for the experiments to be completed in the subsequent laboratory sessions.

Cleaning the tissue culture hood
The next group who uses your hood should find the surfaces wiped down and free of equipment.

1. Aspirate any remaining cell suspensions.
2. Dispose of all vessels that held cells in the biohazard waste box and be sure that all sharps are in the sharps jar.
3. Remove any equipment or supplies that you transferred into the hood and return to the appropriate location.
   • Please leave the equipment that was already there.
4. Spray the TC hood surface with 70% ethanol and wipe with paper towels.
   • Be sure the paper towels are disposed of in the biohazard waste box!
5. Empty the benchtop biohazard bucket into the biohazard waste box.

Part 2: Practice RNA-seq data analysis methods

In preparation for analyzing the RNA-seq data for your Mod 2 research project, you will familiarize yourself with R, a programming language for statistical computing and graphics. RStudio is a free and open-source integrated development environment (IDE) for R. Today you will use 'practice data' to explore the tools offered by RStudio.

R and RStudio are available to you for to use from the Cloud for this class. In this, you can use your personal computers to complete the exercises for this module. In addition, this allows you to work on your analysis outside of the teaching laboratory.

Complete Exercise #1 developed by Amanda Kedaigle, Anne Shen & Prof. Ernest Fraenkel.

Part 3: Discuss research paper

When not in the tissue culture room, you will discuss the research paper by Wei et al. (linked here) with the teaching faculty. In their research, the authors completed examined the effects of etoposide on MCF7 cells, generated from human breast adenocarcinoma. In this article the relationship between DNA damage response (induced by etoposide) and transcription (measured via RNA-seq) is examined.

Our paper discussion will assist you in writing a cohesive story that clearly reports the data and provides strong support for the conclusions made about the data.

During the paper discussion, everyone is expected to participate - either by volunteering or by being called upon!

Introduction

Remember the key components of an introduction:

• What is the big picture?
• Is the importance of this research clear?
• Are you provided with the information you need to understand the research?
• Do the authors include a preview of the key results?

Results

Carefully examine the figures. First, read the captions and use the information to 'interpret' the data presented within the image. Second, read the text within the results section that describes the figure.

• Do you agree with the conclusion(s) reached by the authors?
• What controls are included and are they appropriate for the experiment performed?
• Are you convinced that the data are accurate and/or representative?

Discussion

Consider the following components of a discussion:

• Are the results summarized?
• Did the authors 'tie' the data together into a cohesive and well-interpreted story?
• Do the authors overreach when interpreting the data?
• Are the data linked back to the big picture from the introduction?

Reagents

From Thermo Fisher:

• DLD-1

Media components from Life Technologies, Inc. (unless noted otherwise):

• DLD-1 cell media
  • RPMI 1640
  • 10% fetal bovine serum (FBS)
  • 100X antibiotic solution from Cellgro
    • 10,000 U/mL Penicillin
    • 10,000 U/mL Streptomycin
• phosphate-buffered saline (PBS)
• 0.05% trypsin/0.91 mM EDTA
• trypan blue (Sigma)
• Incubator maintains 37°C, 5% CO2 and 95% relative humidity

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