Difference between revisions of "20.109(S20):Treat cells with etoposide and analyze RNA-seq data (Day2)"

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In this module, you will assess gene expression using two methods: quantitative PCR (qPCR) and RNA-seq.  For timing reasons you will not be able to prepare and submit your treated samples for RNA-seq.  Instead you will use RNA-seq data generated by the teaching faculty.  You will, however, complete the same procedure used by the teaching faculty to prepare the RNA samples and then use your samples in a qPCR experiment to confirm the RNA-seq results.
 
In this module, you will assess gene expression using two methods: quantitative PCR (qPCR) and RNA-seq.  For timing reasons you will not be able to prepare and submit your treated samples for RNA-seq.  Instead you will use RNA-seq data generated by the teaching faculty.  You will, however, complete the same procedure used by the teaching faculty to prepare the RNA samples and then use your samples in a qPCR experiment to confirm the RNA-seq results.
  
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To begin your assay, you will first induce DNA damage by incubating your cells with etoposide.  Etoposide is a chemotherapy drug that generates DNA strand breaks by forming a ternary complex with DNA and topoisomerase II.  This prevents religation of the DNA strands after unwinding.  This effects cancer cells more than non-cancerous cells because cancer cells divide much faster.  We will use etoposide to damage the DNA then examine the differences in gene expression of DLD-1 compared to BRCA2-/- cells.
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To begin your assay, you will first induce DNA damage by incubating your cells with etoposide.  Etoposide is a chemotherapy drug that generates DNA strand breaks by forming a ternary complex with DNA and topoisomerase II.  This prevents religation of the DNA strands after unwinding.  This effects cancer cells more than non-cancerous cells because cancer cells divide much faster.  We will use etoposide to damage the DNA then examine the transcriptome of DLD-1.
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To check that DNA damage was induced by the etoposide treatment, you will measure gene expression of a DNA cell cycle checkpoint factor using quantitative PCR (qPCR).  In qPCR, the amount of a specific transcript can be measured.  The presence of checkpoint factors in an indication of DNA damage in that the cell cycle is halted when excessive damage is incurred by a cell.  For your experiments, the results of p21 expression levels will serve as a control that DNA damage was indeed induced following etoposide treatment.  In addition, the qPCR results will be compared to the expression levels measured via RNA-seq.
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==Protocols==
 
==Protocols==
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===Part 1: Induce DNA damage===
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===Part 1: BE Communication Lab workshop===
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In this exercise, you will induce DNA damage in cells that were seeded by the teaching faculty (using the cells you seeded during the previous laboratory session). Next class you will purify RNA from your cells for quantitative PCR analysis. Control, no treatment flasks will be taken care of by instructors.
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Our communication instructors, Dr. Sean Clarke and Dr. Prerna Bhargava, will join us today for a workshop on preparing and organizing your Journal Club presentations.
  
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[[Image:Sp18 20.109 M2 qPCR overview.png|thumb|750px|center|]]
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===Part 2: Treat cells with etoposide===
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In this exercise, you will induce DNA damage in cells that were seeded by the teaching faculty (using the cells you seeded during the previous laboratory session). Next class you will purify RNA from your cells for quantitative PCR analysis. Control, no treatment flasks will be prepared by instructors.
  
 
#Prepare your working space within the tissue culture hood.
 
#Prepare your working space within the tissue culture hood.
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#*Determine the volume of etoposide stock (100 mM) you need to add to the media for a final concentration of 100 μM in the 10 mL aliquot.
 
#*Determine the volume of etoposide stock (100 mM) you need to add to the media for a final concentration of 100 μM in the 10 mL aliquot.
 
#*Prepare your media +etoposide.
 
#*Prepare your media +etoposide.
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#Retrieve two T75 flasks (one DLD-1 culture and one BRCA2-/- culture) from the 37 °C incubator and visually inspect your cells with a microscope.
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#Retrieve one T75 flask of DLD-1 cells from the 37 °C incubator and visually inspect your cells with a microscope.
 
#*Record your observations concerning media color, confluency, etc. in your laboratory notebook.
 
#*Record your observations concerning media color, confluency, etc. in your laboratory notebook.
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#Move your flasks into the tissue culture hood.
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#Move your flask into the tissue culture hood.
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#Aspirate the spent media from each flask.
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#Aspirate the spent media from the flask.
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#*Be careful not to cross-contaminant between flasks with different cell lines!
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#Add 5 mL of PBS to the flask and rock the plate gently to wash the cells.
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#Add 5 mL of PBS to each flask and rock the plate gently to wash the cells.
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#Aspirate the PBS from each flask.
 
#Aspirate the PBS from each flask.
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#*Again, be careful not to cross-contaminate.
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#Add 10 mL of the media containing etoposide that you prepared in Step #2 to the flask.
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#Add 5 mL of the media containing etoposide that you prepared in Step #2 to the flasks.
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#Carefully move the 'etoposide treated' cells to the 37 °C incubator for 60 min.
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#Carefully move the 'etoposide treated' flasks to the 37 °C incubator for 60 min.
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#When you are done, return to the main laboratory space.
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#Assist your partner if they are still working.  When you are both done, return to the main laboratory space.
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#When the 60 min incubation is complete, return to the tissue culture space and retrieve your flask from the 37 °C incubator.
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#Return to the tissue culture space and retrieve your flasks from the 37 °C incubator.
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#Aspirate the media containing etoposide from the flask.
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#Aspirate the media containing etoposide from each flask.
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#Add 10 mL of fresh media to the flask.  
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#*Be mindful of cross-contamination.
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#Label the flask 'DLD-1 +etop' to denote that the cells were treated with etoposide.   
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#Add 10 mL of fresh media to each flask.
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#*Move the flasks to the 37 °C incubator.
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#Label the flasks containing the DLD-1 and one BRCA2-/- cultures that were treated with etoposide  with '+etop' to denote that the cells were treated with only etoposide.   
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#Clean the hood and return the main laboratory space.
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#*Move these flasks to the 37 °C incubator.
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#Assist your laboratory partner, if necessary, then clean the hood and return the main laboratory space.
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===Part 2: Discuss research paper===
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When not in the tissue culture room, you will discuss the research paper by Dietlein ''et al.'' ([[Media:M2D7 Cancer Discovery-2014-Dietlein-592-605.pdf |linked here]]) with the teaching faculty.  In their research, the authors completed a screen to examine 1319 cancer-associated genes from 67 cell lines to identify cancer-cell specific mutations that are associated with DNA-PKcs dependence or addiction.  Refer to the [[20.109(S17):Module_2#Overview| M2 overview]] for more information on addiction in cancer cells.
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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. 
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<font color=red>'''During the paper discussion, everyone is expected to participate - either by volunteering or by being called upon!'''</font color>
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'''Introduction'''
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Remember the key components of an introduction:
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*What is the big picture?
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*Is the importance of this research clear?
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*Are you provided with the information you need to understand the research?
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*Do the authors include a preview of the key results?
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'''Results'''
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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.
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*Do you agree with the conclusion(s) reached by the authors?
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*What controls are included and are they appropriate for the experiment performed?
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*Are you convinced that the data are accurate and/or representative?
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'''Discussion'''
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===Part 3: Analyze RNA-seq data===
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Today you will analyze the RNA-seq data gathered from untreated DLD-1 and etoposide-treated DLD-1 cells.  Following RNA purification, the samples were submitted to the BioMicro Center for Illumina sequencing.  Illumina sequencing technology,  or sequencing by synthesis (SBS), is used for massively parallel sequencing with a proprietary method that detects single bases as they are incorporated into growing DNA strands.
  
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Consider the following components of a discussion:
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Complete Exercise #2 developed by Amanda Kedaigle, Anne Shen & Prof. Ernest Fraenkel.
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*Are the results summarized?
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*Did the authors 'tie' the data together into a cohesive and well-interpreted story?
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*Do the authors overreach when interpreting the data?
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*Are the data linked back to the big picture from the introduction?
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==Reagents==
 
==Reagents==

Latest revision as of 19:56, 10 March 2020

20.109(S20): Laboratory Fundamentals of Biological Engineering

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Spring 2020 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Screening ligand binding        2. Measuring gene expression        3. Engineering antibodies              


Introduction

In this module, you will assess gene expression using two methods: quantitative PCR (qPCR) and RNA-seq. For timing reasons you will not be able to prepare and submit your treated samples for RNA-seq. Instead you will use RNA-seq data generated by the teaching faculty. You will, however, complete the same procedure used by the teaching faculty to prepare the RNA samples and then use your samples in a qPCR experiment to confirm the RNA-seq results.

To begin your assay, you will first induce DNA damage by incubating your cells with etoposide. Etoposide is a chemotherapy drug that generates DNA strand breaks by forming a ternary complex with DNA and topoisomerase II. This prevents religation of the DNA strands after unwinding. This effects cancer cells more than non-cancerous cells because cancer cells divide much faster. We will use etoposide to damage the DNA then examine the transcriptome of DLD-1.

Protocols

Part 1: BE Communication Lab workshop

Our communication instructors, Dr. Sean Clarke and Dr. Prerna Bhargava, will join us today for a workshop on preparing and organizing your Journal Club presentations.

Part 2: Treat cells with etoposide

In this exercise, you will induce DNA damage in cells that were seeded by the teaching faculty (using the cells you seeded during the previous laboratory session). Next class you will purify RNA from your cells for quantitative PCR analysis. Control, no treatment flasks will be prepared by instructors.

  1. Prepare your working space within the tissue culture hood.
  2. Calculate the volume of etoposide stock needed for DNA damage induction.
    • Obtain an aliquot of pre-warmed media from the 37 °C water bath.
    • Transfer 10 mL of the media into a 15 mL conical tube.
    • Determine the volume of etoposide stock (100 mM) you need to add to the media for a final concentration of 100 μM in the 10 mL aliquot.
    • Prepare your media +etoposide.
  3. Retrieve one T75 flask of DLD-1 cells from the 37 °C incubator and visually inspect your cells with a microscope.
    • Record your observations concerning media color, confluency, etc. in your laboratory notebook.
  4. Move your flask into the tissue culture hood.
  5. Aspirate the spent media from the flask.
  6. Add 5 mL of PBS to the flask and rock the plate gently to wash the cells.
  7. Aspirate the PBS from each flask.
  8. Add 10 mL of the media containing etoposide that you prepared in Step #2 to the flask.
  9. Carefully move the 'etoposide treated' cells to the 37 °C incubator for 60 min.
  10. When you are done, return to the main laboratory space.
  11. When the 60 min incubation is complete, return to the tissue culture space and retrieve your flask from the 37 °C incubator.
  12. Aspirate the media containing etoposide from the flask.
  13. Add 10 mL of fresh media to the flask.
  14. Label the flask 'DLD-1 +etop' to denote that the cells were treated with etoposide.
    • Move the flasks to the 37 °C incubator.
  15. Clean the hood and return the main laboratory space.

Part 3: Analyze RNA-seq data

Today you will analyze the RNA-seq data gathered from untreated DLD-1 and etoposide-treated DLD-1 cells. Following RNA purification, the samples were submitted to the BioMicro Center for Illumina sequencing. Illumina sequencing technology, or sequencing by synthesis (SBS), is used for massively parallel sequencing with a proprietary method that detects single bases as they are incorporated into growing DNA strands.

Complete Exercise #2 developed by Amanda Kedaigle, Anne Shen & Prof. Ernest Fraenkel.

Reagents

  • etoposide, stock = 100 mM (Sigma-Aldrich)

Navigation links

Next day: Purify RNA from etoposide-treated cells and generate cDNA

Previous day: Prepare cells for RNA purification and practice RNA-seq data analysis methods
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