TITLE: Wisecaver Lab algal RNA extraction protocol using Ambion TRI Reagent
AUTHORS: Robert  Auber, Grace   Estep, Jennifer H Wisecaver

[DESCRIPTION]
Modified protocol for extracting RNA from the haptophyte Prymnesium parvum using Ambion Tri Reagent.
Original protocol with toubleshooting  .



[STEPS]
SECTION: Homogenization and RNA extraction (ALL WORK DONE IN THE CHEMICAL FUME HOOD)
11. Warm the frozen centrifuge tube by cupping the area around the pellet in the palm of your hand. As soon as you see the edges of the pellet beginning to melt, add 1 mL  TRI Reagent. Pipet up and down to dislodge pellet, and transfer to a 1.5 mL centrifuge tube. 

All liquid TRI Reagent and chloroform liquid waste should be ejected or decanted into the amber bottle labeled 'Phenol:Chloroform Waste' inside the fume hood. Any plasticware that comes into contact with either of these chemicals needs to be properly disposed of in the sharps bin inside the fume hood labeled 'Mutagen/Carcinogen Sharps Do Not Autoclave'.
SECTION: Homogenization and RNA extraction (ALL WORK DONE IN THE CHEMICAL FUME HOOD)
14. Add 200 µL chloroform. Make sure the cap is closed securely, and mix well by vigorously shaking the tube for 15 s.

SECTION: Homogenization and RNA extraction (ALL WORK DONE IN THE CHEMICAL FUME HOOD)
16. Centrifuge at 12000 x g, 15 min, 4 °C, then transfer approximately 500 µL of the aqueous phase to a fresh 1.5 mL tube. Pipet slowly from the top, being careful not to disrupt the interphase. 

RNA remains exclusively in the aqueous phase whereas DNA and protein are in the interphase and organic phase. Centrifugation at temperatures > 8°C may cause some DNA to partition in the aqueous phase.
SECTION: Pellet Cells
8. Collect approximately 10 million cells growing exponentially in a centrifuge tube and centrifuge 4000 x g, 10 min, 4 °C.
SECTION: Pellet Cells
9. Pour off the supernatant into a P. pavum waste container, and flash freeze the cell pellet in liquid nitrogen. 
SECTION: Pellet Cells
10. Immediately add the TRI Reagent (below) or store in -80 °C freezer. 

It's important to work quickly here as the liquid nitrogen will have lysed many cells already. Material will be highly susceptible to RNAse if allowed to sit at room temperature. 

SECTION: Homogenization and RNA extraction (ALL WORK DONE IN THE CHEMICAL FUME HOOD)
12. Incubate for 5 min  at room temperature.
SECTION: Homogenization and RNA extraction (ALL WORK DONE IN THE CHEMICAL FUME HOOD)
15. Incubate at room temperature for 15 min. 
SECTION: Homogenization and RNA extraction (ALL WORK DONE IN THE CHEMICAL FUME HOOD)
13. OPTIONAL
If you notice a lot of debris following incubation, centrifuge at 12000 x g, 15 min, 4 °C, and transfer the supernatant to a fresh 1.5 mL tube. 

This optional centrifugation is only rarely required to remove insoluble material from homogenates that contain high amounts of protein, polysaccharide, or extracellular material. Centrifugation pellets extracellular membranes, polysaccharides, and high molecular weight DNA, leaving the RNA in the supernatant. With the cell concentrations and species that we are working, this step is typically skipped.
SECTION: RNA Precipitation (ALL WORK DONE AT THE RNA CLEAN WORK STATION)
17. Add 500 µL of isopropanol, and vortex for 10 s. The clear solution should turn cloudy. 

SECTION: RNA Precipitation (ALL WORK DONE AT THE RNA CLEAN WORK STATION)
18. Using the Eppendorf ThermoMixer, incubate at 4 °C  for 30 min.
SECTION: RNA Precipitation (ALL WORK DONE AT THE RNA CLEAN WORK STATION)
19. Centrifuge at 12000 x g, 8 min, 4 °C. Orient the centrifuge tube so that cap hinge is on the outer edge of the rotor. 

Proper orientation of the tube helps to avoid the pelleted RNA when removing the supernatant in subsequent steps. Even if you can't see the pellet, you will know that it is located on the on the bottom corner under the hinge. 
SECTION: RNA Precipitation (ALL WORK DONE AT THE RNA CLEAN WORK STATION)
20. Discard the supernatant by taking off the initial volume with a 1 mL pipette tip. Use a 200 mL pipette tip to get the last bit of liquid. 

You may not see a pellet at this stage, but that is okay. Just be sure to avoid the hinge side of the tube. 

This is the last supernatant and plastic waste that needs to be disposed of in the phenol:chloroform waste. All waste in the remaining steps can go in the standard lab 'look-alike' waste bin. 
SECTION: RNA Precipitation (ALL WORK DONE AT THE RNA CLEAN WORK STATION)
21. Add 1 mLof 75% ethanol to wash the pellet. 

You do not need to vortex or disrupt the pellet at this stage. 

SECTION: RNA Precipitation (ALL WORK DONE AT THE RNA CLEAN WORK STATION)
22. Centrifuge at 7500 x g, 5 min, 4 °C. Again orienting the tube hinge so that it is on the outside of the rotor. 

SECTION: RNA Precipitation (ALL WORK DONE AT THE RNA CLEAN WORK STATION)
23. Discard the supernatant by taking off the initial volume with a 1 mL pipette tip. Briefly spin down the tube using the little bench top spinner, and then use a 200 mL pipet tip to get the last bit of liquid. 

Again, even if you can't see the pellet, you will know that it is located on the on the bottom corner under the hinge. So be careful to avoid that side of the tube. Pipette away the supernatant by tipping the tube slightly towards you and pipetting the liquid from the bottom corner opposite the hinge. 
SECTION: RNA Precipitation (ALL WORK DONE AT THE RNA CLEAN WORK STATION)
24. Air dry the RNA pellet for 3 min by placing the tube on its side inside a folded kim wipe. 

Do not overdry the RNA pellet as this will greatly decrease its solubility. 

Two major sources of RNase contamination are fingers and dust. Never leave the tube open and oriented up for dust to get into your sample. 
SECTION: RNA Precipitation (ALL WORK DONE AT THE RNA CLEAN WORK STATION)
25. Re-suspend the pellet in 50 µL of nuclease-free water. Pipette up and down 10 times letting the water wash down the hinge side of the tube. 
SECTION: QC and clean up
26. Place a 5 mL aliquot of your RNA sample into a separate 1.5 mL tube for QC and store on the Eppendorf ThermoMixer at 4 °C. Store the rest of your sample immediately in the -80 °C freezer. 

SECTION: QC and clean up
27. Make sure all TRI Reagent and chloroform waste is properly disposed of in the chemical fume hood. 
Place all RNA clean pipettes, racks and consumables (tips, tubes, water etc) back in the RNA clean drawer. 
Wipe down the chemical fume hood and RNA clean workstation with 75% ethanol. 


SECTION: Prepare Station and Materials
1. Set a large bench top centrifuge with 50 mL falcon tube compatibility, and a ThermoMixer to 4 °C.
SECTION: Prepare Station and Materials
5. Prepare 75% ethanol. Per sample, mix 1.5 mL ethanol with 500 µL nuclease free water. Scale up for how ever many samples you are processing. 


SECTION: Prepare Station and Materials
3. Wipe down with RNAse away:
Chemical fume hood. Remove the spill pad from the chemical fume hood if present and replace with a new pad. 
RNA clean work station
Rotors on large microcentrifuge and bench top spinner
ThermoMixer
Plastic racks (in RNA clean drawer)
Pipettes (in RNA clean drawer)
SECTION: Prepare Station and Materials
6. Using gloved hands freshly sprayed with RNAse away, get three 1.5 mL microcentrifuge tubes per sample from the bag in the RNA clean drawer. Cap each tube tightly and place the sealed bag back in the clean drawer. Label each tube vertically on the side with the sample's unique identifier. Write down the sample ID and description in your lab notebook. 

The identifier should start with a letter and be no more than 12 characters long. Write it carefully using a fine tip permanent marker as one of these tubes will contain your final sample that will be shipped off for RNA sequencing. 
SECTION: Prepare Station and Materials
2. Get liquid nitrogen if starting from live cells.
SECTION: Prepare Station and Materials
4. Get RNA clean tips out from the clean drawer. Place one set of 1 mL pipettes and tips inside the chemical fume hood. Place one set of 50 mL and 1.5 mL tube racks in the chemical fume hood. 
SECTION: Prepare Station and Materials
7. Place two labeled 1.5 mL tubes per sample inside the chemical fume hood. Leave the third tube at the RNA clean workstation.