Total RNA Extraction Protocol

Notes:

The MJ Research PTC-200 Thermal Cycler was used for all reactions. A heated lid was used to minimize evaporation on long incubations. This machine can easily obtain temperatures from 4-100 °C and has a very rapid ramp time. The procedures that require removal to ice were simply allowed to drop to 4°C.

Sample Preparation

Partition

• In a 2 ml screw cap tube with o-ring:
• Add 250 uL Resuspended Cells in PBS
• Add 750 uL TRIzol LS (1 ml/ 107 cells)
• Add 800 uL Chloroform
• Shake vigorously for 15 seconds
• Let stand for 10 min
• Centrifuge at 4 °C for 15 minutes, 12,000 x g
• Aliquot the top aqueous layer into a new 1.5 ml tube. (The DNA is in between the two phases; do not take this white precipitate.)

Precipitation

• Add 1 uL glycogen or NF pellet paint (Novagen)
• Add equal volume 2-Propanol
• Invert to mix the layers
• Let stand for 10 min
• Centrifuge at 4 °C for 15 minutes, 12,000 x g
• Carefully remove supernatant and then quick spin
• Remove as much 2-Propanol as possible with P10 Tip

Wash pellet

• Add 1 mL 70-75% ethanol
• Centrifuge at 4 °C for 15 minutes, maximum speed
• Carefully remove supernatant and then quick spin
• Remove as much ethanol as possible with P10 Tip
• Air dry in the hood for 10 minutes

Resuspend

• Add water (amount varies with yield)
• Quick vortex to mix
• Heat 60 °C for 10 minutes
• Can be stored at -80 °C

Quantitate the RNA yield

• Dilute samples for Optical Density reading in a 0.5 ml tube
• Add 1 uL sample
• Add 79 uL 1X TE
• Measure and calculate the following:
  • A260
  • A280
  • Concentration
  • Purity
  • Dilution factor ( (sample + water)/sample )
  • Stock concentration (concentration * dilution factor)
  • Total volume (total volume * sotck concentration)
  • Yield