RNA Preparation Protocol

Part 1: Harvesting Cells

Materials and Reagents:

• Phosphate Buffered Saline (PBS)
• Trypsin
• 5 mL sterile pipets
• Vacuum apparatus w/ autoclaved Pasteur pipets.
• Eppendorf Tubes
• Tri-Reagent

Procedure:

1. Prepare Biosafety cabinet as explained in procedure 2, steps 1-2.
2. Vacuum media from all wells.
3. Wash each well with 1 mL PBS (or 0.5 mL for 24-well plates). Vacuum PBS.
4. Add 200 µL Trypsin. Wait 10-15 minutes until cells are fully trypsinized.
5. Add 800 µL PBS (300 uL for 24-well plates). Pipet each well’s contents into marked Eppendorf tubes.
6. Centrifuge tubes.
7. Aspirate PBS.
8. Add 0.5 mL TRI-Reagent to each Eppendorf tube. Freeze.

Part 2: RNA Isolation

Materials and Reagents:

• Centrifuge
• BCP
• Isopropanol
• 75% ethanol
• Nuclease free H2O
• Eppendorf tubes
  

Procedure:

1. Remove cells from freezer and let thaw at room temperature.
2. Add 50 µL BCP to each Eppendorf tube. Mix well. Let sit for 5-15 minutes.
3. Centrifuge at 12x g for 10-15 min at 4 °C.
4. Transfer aqueous phase (top, clear layer) to a new tube. Store the pink Tri-Reagent phase for later protein analysis.
5. Add 250 µL isopropanol. Vortex for 5-10 sec. Let sit for 5-10 min.
6. Centrifuge at 12x g for 8 min at 4 °C.
7. Discard supernatant.
8. Add .5 mL 75% ethanol.
9. Centrifuge at 7.5x g for 5 min.
10. Discard ethanol and briefly air dry RNA pellet.
11. Dissolve RNA pellet in 25 µL Nuclease-free water. Freeze

Part 3: cDNA Reverse Transcription

Materials and Reagents:

• Nuclease-free water
• cDNA Reverse Transcriptase reagents
• 8-strip PCR tubes
• Micropipeters
• Eppendorf Tubes
• ProFlex PCR System

Procedure:

1. Make up Master Mix with no water.

   1. Per Reaction:
      
      1. 2uL Buffer
      2. 2uL Primers
      3. 1uL Reverse Transcriptase (red cap)
      4. 0.8uL dNTP (purple cap)
      5. 4.2uL Nuclease Free Water
2. Combine 10 µL Master Mix and 10 µL RNA in each PCR tube.
3. Briefly vortex PCR tubes, then mini-centrifuge them for 10-15 sec.
4. Place in ProFlex PCR system. Initiate “High Capacity cDNA Reverse Transcription.” Freeze.

Part 4: Real-Time PCR

Materials and Reagents:

• 96-well Real-Time PCR plates
• PCR plate covers
• Water
• PCR probes/primers
• 2x TaqMan
• Real-Time PCR Machine

Procedure:

1. Determine how many genes you need to test for (PPIA, Nkx6.1, Fos, etc). Repeat the following steps for each probe.
2. Dilute cDNA with Nuclease Free H2O so that you have 10 ul for each probe you will test.  Mix thoroughly, centrifuge for 10-15 sec
3. If you have a primer with both forward and reverse primers separated, you will need to make the mixture you will use as your primer: - 50 ul forward - 50 ul reverse - 400 ul IDTE buffer

4. Make a Master Mix. Multiply the volume of each reagent by 4n where n is the number of samples you have. Mix well.

   1. Taqman (thin tubes)

      1. 2x Taqman - 5uL
      2. Primer - .5uL
      3. H20 - 2uL

   2. SyberGreen

      1. Sybergreen - 5uL
      2. Primer - .5uL
      3. H2O - 2uL                             
5. Get out a sufficient number of PCR tubes to run your samples in.
6. Pipet 26.25 µL Master Mix to each PCR tube.
7. Pipet 8.75 µL cDNA to each PCR tube with multichannel pipet. Mix.
8. Pipet 10 µL Master Mix + cDNA to 96 well PCR plate in triplicate.
9. After you have completed steps 3-7 for each probe, attach cover to PCR plate. At this point you can freeze plate for later, or run it in the Real-Time PCR machine.
10. Spin PCR plate in plate spinner.
11. Place carefully in Real-Time PCR machine.
12. Open the StepOne program on the computer.
13. Select “Advanced Setup.”
14. Set up Experimental Properties.
15. Go to Plate Setup.
16. Go to Assign Targets and Samples.
17. Go to Run Method. Verify that Reaction Volume Per Well is 10 µL.

Go to Run. Start Run!

Supplemental Materials:

[.pdf] Essentials of RT-PCR (Life Technologies)[.pdf] TaqMan Universal Master Mix Protocol
Tri-Reagent-Protocol.pdf