20.109(S17):Purify RNA for quantitative PCR assay and treat cells for survival assay (Day3)

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20.109(S17): Laboratory Fundamentals of Biological Engineering

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Schedule Spring 2017        Announcements        Assignments        Homework        Communication
       1. High-throughput ligand screening        2. Gene expression engineering        3. Biomaterials engineering              

Introduction

Differential gene expression is used to translate information perceived by a cell into a gene product. All forms of life rely on gene expression to generate proteins and non-protein coding gene products that provide the machinery for cell survival. In this, gene expression is the most basic mechanism by which genotype results in a phenotype. One method researchers use to examine gene expression is to quantify transcript levels for specific genes.

In this module, we are interested in the effect of DNA damage on cell survival and gene expression. To address these questions we are using a BRCA2- mutant cell line. When DNA damage is induced in these cells (either naturally or with the chemical etoposide), the absence of BRCA2 results in a dependence or addiction to the NHEJ pathway. If a drug is introduced following DNA damage that inhibits NHEJ repair, the expected outcome is decreased cell survival compared to wild-type cells; hence the inclusion of the cell survival assay in this module.

We will use two experimental approaches to evaluate gene expression: qPCR and RNA-seq. Though the intricacies of each method will be discussed on D4 and D7, respectively, a critical first step for both is the purification of high-quality RNA. RNA purification is complicated by ribonuclease enzymes, such as RNase. RNases are ubiquitous can be difficult to neutralize. Furthermore, RNases are present in the cells and tissues from which the RNA is isolated. The 'gold standard' for RNA purification is an organic extraction method. In this process a sample is homogenized in a phenol solution then centrifuged, which separates the sample into three phases: a lower organic layer, a denatured protein and gDNA middle layer, and an RNA upper layer. The RNA from the upper layer is alcohol precipitated and rehydrated. Because this method is labor-intensive and requires chlorinated organic reagents, researchers often use column-based purification kits.

We will use a commercially available column-based RNA purification system. Though the reagents are proprietary, the manufacturer provides some details concerning the inner workings of the kit. The key component is a silica-based membrane that binds RNA molecules longer that 200 nucleotides, which enriches for messenger RNA (mRNA) molecules. Prior to RNA isolation using the membrane, cells are lysed in the presence of a guanidine-thiocyanate-containing buffer that inactivates the RNases naturally present in the sample. Then ethanol is added to the lysate to promote binding of the RNA to the silica.

Following RNA purification, you will generate cDNA to use in your quantitative PCR analysis. Complementary DNA, or cDNA, refers to DNA that is generated from RNA. The ability to natively generate cDNA is unique to retroviruses. In research, this technology is available as a tool because the reverse transcriptase enzyme can be purified and used in exogenous reactions. We will generate cDNA from purified RNA to study gene expression as DNA molecules are more stable and, therefore, easier to work with experimentally. As with the RNA purification, we will use a kit to generate cDNA. The kit includes the necessary buffers and reverse transcriptase enzyme. First, the RNA is combined with a poly-T primer and oligonucleotides, then incubated at a high temperature for a denaturation step. An RNase inhibitor, reverse transcriptase, and buffers are then added. This mix is incubated at a temperature optimal for the reverse transcriptase enzyme to generate cDNA. The specific enzyme we will use is SuperScript III RT, and like the buffers included in the RNA purification kit, the genetic manipulations used to modify this reverse transcriptase enzyme are proprietary.

Protocols

Part 1: Finish Western blot analysis

  1. Obtain your blots from the front bench. Save the primary antibody solution by pouring it into a conical tube, writing the identity of the antibodies and the date on the tube.
    • Because the antibody is in excess, the diluted primary solution may be re-used on another blot and is thus worth saving until you see your Western blot.
  2. Add enough TBST to cover your membrane - no need to measure a volume.
    • Keep in mind that the washing steps work by dilution, so it is a balance between adding enough to create a sink for the primary antibody, but not so much that you make a huge mess on the shaker!
    • TBST is Tris-buffered saline with 0.1% Tween 20 (a surfactant).
  3. Shake your container for 5 min at 80 rpm using the room temperature shaker.
  4. Repeat for a total of 4 washes.
  5. Just before pouring off the last wash, prepare the secondary antibodies.
  6. Dilute the secondary antibodies in 10 mL of Blocking Buffer.
    • They are light sensitive so find them on the front bench next to the Blocking Buffer and then wrap your tube in aluminum foil.
    • For α-tubulin (mouse primary antibody), use the goat anti-mouse IR800 (GREEN) antibody at 1:10,000; and for BRCA2 (rabbit primary), use donkey anti-rabbit IR680 (RED) at 1:10,000.
  7. After the last wash, add your secondary antibody solution, place on the room temperature shaker, and cover your Western blot container with aluminum foil.
  8. Shake at 65 rpm for 60 min.
    • During this incubation, half of the class will begin Part 2 and half of the class will begin Part 3, according to the teaching faculty instructions.
  9. Pour off the secondary antibody in the sink.
  10. Wash the membrane by adding TBST and shake for 5 min at 80 rpm, using the room temperature shaker.
  11. Repeat for a total of 4 washes.
  12. The Odyssey scanner is located in the Lauffenburger laboratory (56-378), one of the teaching faculty will accompany you there in groups to scan your blots.

Part 2: Prepare samples for quantitative PCR assay

You will move your cells to the main laboratory to purify the RNA from your cells. Before you retrieve your flasks from tissue culture, prepare your laboratory bench for work with RNA. As noted in the introduction, RNA is very sensitive to degradation and caution must be taken to preserve your sample.

First, obtain a piece of absorbent paper. This will be your RNAse free work space. Spray the absorbent paper with RNase Away and dry with a Kimwipe. Perform this cleaning procedure with all of the equipment you will need for the RNA purification protocol (read through Part 2a Step #11 to the end of the protocol). Also, obtain all of the aliquots you will need from the front laboratory bench and clean before placing the tubes on the absorbent paper work space. Lastly, obtain a 50 mL conical tube and label it 'waste'.

Sp17 20.109 RNA purification R2.png

Part 2a: Purify RNA from cells

  1. Prepare your working space at your lab bench.
  2. Retrieve your four T75 flasks (DLD-1, BRCA2-, DLD-1 +etop, and BRCA2- +etop) 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.
  3. At your laboratory bench, aspirate the media from each flask.
  4. Wash the cells by adding 5 mL PBS using a 5 mL pipet. Slightly tip the flask back and forth to rinse the cells then aspirate the PBS by covering the pasteur pipet attached to the aspirator with a yellow tip.
    • Be sure to use a fresh tip for each flask.
  5. With a 2 mL pipet, add 2 mL of trypsin to each flask.
  6. Tip the flask in each direction to distribute the trypsin evenly then incubate the cells at 37 °C incubator for 5 minutes using a timer.
  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, add 3 mL of PBS to each flask.
    • To ensure you collect all of the cells in your flask, pipet the PBS down the bottom of the flask to wash the cells from the surface.
    • Pipet up and down 3 times.
    • 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. Centrifuge your suspensions at 1000 rpm for 5 min to pellet the cells.
  11. Carefully remove your tubes from the centrifuge and return to your laboratory bench.
    • Avoid agitating your tubes as the pellet is easily disrupted.
  12. Slowly pour the supernatant from your tubes into your 50 mL waste conical tube.
    • Do not shake, tap, or flick the tubes to remove excess liquid as this will disrupt your cell pellet.
  13. Add 350 μL of RLT and pipet up and down to mix.
  14. Transfer the cell / RLT suspension to a Qiashredder column (purple).
    • Be sure to label both the column insert and collection tube.
    • Only 700 μL at a time can be loaded onto the column. If you have more than 700 μL, consult the teaching faculty.
  15. Centrifuge at 16,000 rpm for 2 min.
  16. Remove the columns from the collection tubes and add 350 μL of 70% ethanol, then pipet to mix.
  17. Transfer 700 μL of the RNA / ethanol suspension to an RNAeasy column (pink).
  18. Centrifuge at 10,000 rpm for 15 s then discard the flow-through from the collection tube in your 50 mL waste conical tube.
  19. Repeat Steps #17 - 18 until all of the RNA / ethanol suspension has been passed through the RNAeasy column.
  20. Add 700 μL of RW1 and centrifuge at 10,000 rpm for 30 s, then discard the flow-through in your 50 mL waste conical tube.
  21. Add 500 μL of RPE and centrifuge at 10,000 rpm for 30 s, then discard the flow-through in your 50 mL waste conical tube.
  22. Add 500 μL of RPE and centrifuge at 10,000 rpm for 2 min, then discard the flow-through in your 50 mL waste conical tube.
  23. Move the columns into new collection tubes, then centrifuge at 16,000 rpm for 1 min.
  24. Move the columns into 1.5 mL tubes (from the Qiagen RNeasy Mini Kit).
    • Label the base of the tubes to ensure you keep track of your samples.
  25. Add 30 μL of RNase free water, then centrifuge at 10,000 rpm for 1 min.
  26. Alert the teaching faculty when you are done and you will be escorted to the NanoDrop to check the concentrations of your purified RNA samples.

Return your 50 mL 'waste' conical tube to the front bench. It will be disposed of as hazardous waste.

Part 2b: Generate cDNA

  1. Calculate the volume of your RNA solution that contains 1 μg of RNA.
    • Alert the teaching faculty if this volume is greater than 9 μL.
  2. Add the appropriate volume of DEPC-treated water to a PCR tube.
    • The total volume of RNA + DEPC-treated water should equal 9 μL.
  3. Add 1 μL of the oligo(dT)20 primer from the stock located at the front laboratory bench.
  4. Add the volume of your RNA solution that you calculated in Step #1.
  5. Incubate the RNA-primer mixture in the 65 °C water bath on the front laboratory bench for 5 min.
  6. Retrieve your tube and incubate on ice for 1 min.
  7. Prepare enough 'cDNA Synthesis Master Mix' for 4.5 reactions given the volumes of each component required for one reaction that are listed below. You will pipet the stock reagents at the front laboratory bench using filtered pipet tips.
    • 2 μL of 10X RT buffer
    • 4 μL of 25 mM MgCl2
    • 2 μL of 0.1 M DTT
    • 1 μL of RNaseOUT
    • 1 μL of SuperScript III RT
  8. Add 10 μL of cDNA Synthesis Master Mix to each of you RNA-primer tubes.
  9. Run the 'RT' program on the thermocycler:
    • Reactions will be incubated at 50 °C for 50 min, then terminated at 85 °C for 5 min, and finally held at 4 °C for 2 min.
  10. Retriever your tubes and add 1 μL of RNase H. Incubate in the 37 °C incubator for 20 min.
  11. Your cDNA will be stored in the -20 °C freezer until the next laboratory session.

Part 3: Treat cells for viability assay

Today you will induce DNA damage in DLD-1 and BRCA2- cells seeded by the teaching faculty. Following DNA damage induction, you will treat with an NHEJ-inhibitory drug to evaluate conditional lethality in the BRCA2- cell line. In addition, you will examine the effect of a PARP inhibitor on cell survival.

Compound 401 is a selective, reversible DNA-dependent protein kinase and mTOR inhibitor, a serine/threonine protein kinase. Research conducted in the Samson Laboratory showed that Compound 401 inhibits non-homologous end joining (NHEJ), but not other DNA repair pathways.

Olaparib is a PARP (poly ADP ribose polymerase) inhibitor, an enzyme involved in DNA repair. PARP inhibitors have been shown to kill BRCA2 null cells.

Sp17 20.109 cell survival R2.png
  1. Prepare your working space on your lab bench.
  2. Obtain an aliquot of pre-warmed media containing 100 μM etoposide (9 mL) and plain media (13 mL) from the 37 °C water bath.
  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-contaminate 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. Repeat the PBS wash a total of 2 times.
  9. Add 2 mL of media (without etoposide!) to wells A1, B1, C1 and A3, B3, C3.
  10. Add 2 mL of the media containing etoposide that you prepared in Step #2 to wells A2, B2 and A4, B4.
  11. Carefully put your plate in the 37 °C incubator for 60 min.
  12. Return to the tissue culture space and retrieve your plate from the 37 °C incubator.
  13. Aspirate the wells with media containing etoposide then complete two PBS washes as done above.
    • Be mindful of cross-contamination.
  14. Add 2 mL of fresh media to the wells that contain the etoposide treated cells.
  15. Calculate the volume of drug you need to add to each well for the final concentrations shown on the figure below.
    • Compound 401 (stock concentration = 10 mM)
    • Olaparib (stock concentration = 50 mg/mL, molecular weight 435 g/mol)
  16. Add the appropriate volume of drug to the wells according to the plate map below.
  17. Carefully move your 12-well plate to the 37 °C incubator.
  18. Clean the hood and return the main laboratory space.
Sp17 20.109 M2D3 plate map.png

Reagents

For the Western blot:

  • TBST
    • Tris-buffered saline
    • 0.1% Tween 20
  • rabbit anti-human anti-BRCA2 antibody: gift of the Engelward lab at MIT
  • mouse anti-human anti-tubulin antibody (DM1A, Cell Signaling)
  • goat anti-mouse antibody (Li-COR)
  • donkey anti-rabbit antibody (Li-COR)

For the qPCR sample preparation:

  • RNeasy mini kit (Qiagen)
  • SuperScript III First-Strand kit (synthesis system for RT-PCR by Invitrogen)

For the drug treatment:

  • Etoposide (Sigma Aldrich)
  • Compound 401 (Abcam)
  • Olaparib (Selleck Chemicals)

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