Difference between revisions of "20.109(S18):Induce DNA damage and apply drug treatments for cell viability and identification of regulatory motifs in RNA-seq data (Day8)"

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(Introduction)
(Part 2: Examine qPCR results)
Line 115: Line 115:
 
#*Plot the reads for p21 (also called CDKN1A):   
 
#*Plot the reads for p21 (also called CDKN1A):   
 
#**<code>plotCounts(dds,"CDKN1A", intgroup="group")</code>
 
#**<code>plotCounts(dds,"CDKN1A", intgroup="group")</code>
 +
#*To also plot a heatmap of the expression of CDKN1A and genes targeted by p53:
 +
#**<code>load("~/Desktop/RNA-seq data analysis/afterAnalysis.RData")
 +
#**<code> p53_targets =c('CDKN1A','BTG2','FBXW7','GADD45A','SFN','GTSE1','ZNF385A','PCBP4','GPX1','GPX2','SESN2','ALDH4A1','SOD2','CFLAR','PTGS2','CCNG1','DDR1','HBEGF','PPM1D','MYO6','TNFRSF10D','TNFRSF10B','APAF1','BAX','FAS','PMAIP1','PERP','TP53AIP1','TP53I3','BBC3','SIVA1','PTP4A3','PML','PTPRVP','PIDD1','DDB2','ERCC5','FANCC','XRCC5','MGMT','MLH1','MSH2','RRM2B','POLK','XPC')
 +
mat = assay(rld)[p53_targets, ]
 +
mat = mat - rowMeans(mat)
 +
pheatmap(mat, annotation_col=df)</code>
  
 
===Part 3: Identify regulatory motifs in RNA-seq data===
 
===Part 3: Identify regulatory motifs in RNA-seq data===

Revision as of 19:11, 26 March 2018

20.109(S18): Laboratory Fundamentals of Biological Engineering

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Spring 2018 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Assessing ligand binding        2. Measuring gene expression        3. Engineering biomaterials              


Introduction

As discussed previously, we are using etoposide in our experiments to induce DNA damage. To explore cancer treatment options that exploit pathway dependency, or 'addiction', you will first induce DNA damage with etoposide then use a previously identified inhibitor of NHEJ to 'kill' the BRCA2-/- cells. Ideally, the cancer cells will inquire more loss, or death, than the DLD-1 parental cells.

INCLUDE BACKGROUND INFORMATION FROM DIETLEIN REFERENCE

Each team will select one NHEJ-targeting drug, either mibefradil or loperamide. The mechanism by which mibefradil and loperamide target NHEJ are unknown, though both drugs are useful in the treatment of other conditions:

  • Mibefradil is is a calcium channel blocker used to treat hypertension and chronic angina pectoris.
  • Loperamide is known to slow the contractions of the intestines and is used to treat gastroenteritis, inflammatory bowel disease, and short bowel syndrome.

Protocols

Before you begin, select which drug (mibefradil or loperamide) you will use for your experiments in this module and enter your team information in the table below.

Team Mibefradil Loperamide
T/R
1
2
3
4
W/F
1
2
3
4

Part 1: Induce DNA damage and apply treatments

You will assess cell viability in response to DNA damage and drug treatment to investigate the conditions used when the RNA-seq data was generated. In this exercise, you will induce DNA damage in the cells that were seeded by the teaching faculty then add the drug that you selected on M2D2. Next class you will examine survival of your cells using an assay the abundance of live cells.

  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 (11 mL).
    • Determine the volume of etoposide stock (100 mM) you need to add to the media for a final concentration of 100 μM.
  3. Retrieve your 12-well plates 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 plate into the tissue culture hood.
  5. Aspirate the spent media from each well.
    • Be careful not to cross-contaminant between wells with different cell lines!
  6. Add ~1 mL of PBS to each well and rock the plate gently to wash the cells.
  7. Aspirate the PBS from each well.
    • Again, be careful not to cross-contaminate.
  8. Add 1 mL of PBS to well A1 and A3.
    • These wells will be the 'no DNA damage' controls for the DLD-1 and BRCA2- cells.
  9. Add 1 mL of the media containing etoposide that you prepared in Step #2 to the empty wells.
  10. Carefully put your plate in the 37 °C incubator for 60 min.
  11. Assist you partner if they are still working. When you are both done, return to the main laboratory space.
  12. Return to the tissue culture space and retrieve your plate from the 37 °C incubator.
  13. Aspirate the PBS or media containing etoposide from each well.
    • Be mindful of cross-contamination.
  14. Add 2 mL of fresh media to each well.
  15. Calculate the volume of drug you need to add to each well for the final concentrations shown on the figure below.
    • Be sure to use the concentrations appropriate for the drug you chose.
    • The stock concentrations of mibfradil and loperamide are 10 mM.
    • It may be helpful to dilute the drug stocks to avoid pipetting very small volumes.
  16. Add the appropriate volume of drug and move your 12-well plate to the 37 °C incubator.
  17. Assist your laboratory partner, if necessary, then clean the hood and return the main laboratory space.
Sp17 20109 M2D2 drug plate map.png

Part 2: Examine qPCR results

Before you can apply the statistical tools you learned in Mod1 to your data, you must first normalize the expression levels of your gene of interest and p21. To account for any unintended biases in RNA purification and / or cDNA preparation, it is important to normalize the expression of the transcript of interest to expression of a housekeeping or constitutive gene. Ideally, the gene to which the data of interest are normalized is not responsive to the treatment tested. In our experiment, we used GAPDH because it is not expected to be responsive to etoposide treatment. How might you confirm this assumption?

  1. Review your data posted on the Class data page.
    • Remember that the DLD-1 and BRCA2-/- samples were prepared by the teaching faculty.
    • You prepared your DLD-1 +etoposide, DLD-1 +etoposide +drug, BRCA2-/- +etoposide, and BRCA2-/- +etoposide +drug samples.
    • All samples were probed using the primers you designed to your gene of interest, along with the p21 and GAPDH primers.
    • Each reaction was completed in triplicate. Note: these are technical replicates.
    • The data are represented as the 'threshold cycle' CT or amplification cycle at which SYBR Green fluorescent signal was detected (review M2D5 Introduction).
  2. Normalize your gene of interest and p21 expression to GAPDH expression (ΔCT).
    • Subtract the GAPDH CT value from your gene of interest and p21 CT values using the appropriate treatment conditions, according to the screenshot below.
      Sp17 20.109 M2D9 qPCR normallization.png
  3. Exponentially transform each normalized value to the ΔCT expression.
    • ΔCT expression = 2-ΔCT.
  4. Average the replicates for each treatment, then calculate the 95% CI and t-test p-value.
    • With this information, graph your data with error bars and include information concerning any statistical significance.
  5. Are these results consistent with those from the RNA-seq data for p21 expression?
    • Load the data:
      • load("~/Desktop/RNA-seq data analysis/preprocessed_data.RData")
      • library("DESeq2")
    • Plot the reads for p21 (also called CDKN1A):
      • plotCounts(dds,"CDKN1A", intgroup="group")
    • To also plot a heatmap of the expression of CDKN1A and genes targeted by p53:
      • load("~/Desktop/RNA-seq data analysis/afterAnalysis.RData")
      • <code> p53_targets =c('CDKN1A','BTG2','FBXW7','GADD45A','SFN','GTSE1','ZNF385A','PCBP4','GPX1','GPX2','SESN2','ALDH4A1','SOD2','CFLAR','PTGS2','CCNG1','DDR1','HBEGF','PPM1D','MYO6','TNFRSF10D','TNFRSF10B','APAF1','BAX','FAS','PMAIP1','PERP','TP53AIP1','TP53I3','BBC3','SIVA1','PTP4A3','PML','PTPRVP','PIDD1','DDB2','ERCC5','FANCC','XRCC5','MGMT','MLH1','MSH2','RRM2B','POLK','XPC')

mat = assay(rld)[p53_targets, ] mat = mat - rowMeans(mat) pheatmap(mat, annotation_col=df)

Part 3: Identify regulatory motifs in RNA-seq data

attach exercise...

Reagents

  • etoposide, stock = 100 mM (Sigma-Aldrich)
  • mibefradil dihydrochloride hydrate, stock = 10 mM (Sigma-Aldrich)
  • loperamide hydrochloride, stock = 10 mM (Sigma-Aldrich)

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