TITLE: Brain Tissue RNA Extraction -- University of Minnesota TMCs
AUTHORS: David A Bernlohr, Hector Martell Martinez

[DESCRIPTION]
RNA extraction protocol from snap frozen tissues and/or cell pellets adapted from PureLink RNA Mini Kit and PureLink with TRIzol Reagent protocols (attached). Protocol here used with neurological tissues prior to Bulk RNA sequencing. 

  
  
  


[GUIDELINES]
See Appendix in attached PDF -   


[STEPS]
SECTION: Preparation
1. Choose and locate samples: box year and number, slot number.
SECTION: Preparation
2. Spray benchtop with 70% ethanol or RNase away.
SECTION: Preparation
3. Fill two containers with dry ice. One for the tools in step #4. Second for holding tissue samples.
SECTION: Preparation
4. Wipe cutting board, razor, and forceps all with 70% ethanol. 
 

 

SECTION: Preparation
5. Set (per sample) one lysing tube (i.e., green capped and contains tiny white beads), three 1.5 ml tubes, one spin cartridge with a collection tube, and one collection tube aside. Label.
SECTION: Preparation
6. DNase Preparation (if needed): Resuspend DNase with 550 µl RNase-free water (provided). 
SECTION: Preparation
1.1. Note sample information (e.g., specimen ID, animal ID, genotype, age, sex, etc).
SECTION: Preparation
1.2. Tip: Note the quantity of tissue for each sample. If low, cutting tissue is not needed.
SECTION: Preparation
6.1. Create a master mix. Each sample needs 80 µl
        i. 10X DNase I reaction buffer: 8.0 µl
        ii. Resuspended DNase: 10.0 µl
        iii. RNase-free water: 62.0 µl
SECTION: Homogenize Snap Frozen Tissues
7. Grab TRIzolTM Reagent from the 4°C fridge Place in a fume hood.
SECTION: Homogenize Snap Frozen Tissues
8. Pipette 1000 μl TRIzolTM into each lysing tube in the fume hood. [1]
SECTION: Homogenize Snap Frozen Tissues
9. Cut tissue from each sample (if applicable) and put in the lysing tube. Invert to mix. 
          

SECTION: Homogenize Snap Frozen Tissues
10. Replace the dry ice in the first container with wet ice (optional–only if you wish to cool lysing tubes on wet ice instead of the cold room).
SECTION: Homogenize Snap Frozen Tissues
11. Homogenize all samples in tissue homogenizer. [2] 
SECTION: Homogenize Snap Frozen Tissues
12. If homogenizing more than once, place all lysing tubes on wet ice OR place in a cold room for 2-mins between homogenization runs. Cooling is necessary to prevent RNA degradation from heat produces while the samples were being homogenized. 
SECTION: Homogenize Snap Frozen Tissues
13. Incubate the lysate with TRIzolTM at  for 5 mins.
SECTION: Homogenize Snap Frozen Tissues
9.1. Ensure all tissue is immersed in TRIzol. Each sample has its                             own lysing tube. 
SECTION: Homogenize Snap Frozen Tissues
9.2. Wipe all tools with 70% ethanol before, between, and after touching samples. c. 
SECTION: Homogenize Snap Frozen Tissues
9.3. Tip: Store samples in TRIzolTM at -80°C before homogenizing, if you wish to stop at this step.
SECTION: Homogenize Snap Frozen Tissues
11.1. a. Select tissue type within recommended programs. Most run for 40 seconds. [3] 
SECTION: Homogenize Snap Frozen Tissues
11.2. b. Repeat homogenization until tissue appears a fine powder within the TRIzol liquid.
    i. Tip: The smaller the tissue, the less homogenization needed.     
    For brain regions, one typically will only need one round of 40
    second homogenization. For larger organs (e.g., liver, kidney,
    spleen) or greater tissue quantity, three rounds may be
    necessary.        
    ii. Tip: Pink foam is commonly seen after homogenizing brain
    regions. Foam will disappear in a few minutes. Occurs due to the
    speed the tubes are spinning.
SECTION: Homogenize Cell Pellets
14. Grab TRIzolTM Reagent from the 4°C fridge Place in a fume hood. 

SECTION: Homogenize Cell Pellets
15. Pipette 1000 μl TRIzolTM per 1 million cells into each 1.5 ml tube in the fume hood. [1]
SECTION: Homogenize Cell Pellets
16. Incubate in TRIzolTM for 2-3 mins.
SECTION: Homogenize Cell Pellets
17. Resuspend cell pellets by pipetting up-and-down 15-25 times. Keep consistent between cells.
SECTION: Homogenize Cell Pellets
18. Allow the tubes to sit for 5 minutes.
SECTION: Homogenize Cell Pellets
19. Repeat steps #4-5 for each tube until pellets are dissolved. a. Tip: Store samples in TRIzol at -80°C before homogenizing, if you wish to stop for now.
SECTION: Homogenize Cell Pellets
14.1. Tip: Remove cell pellets from -80°C storage just before beginning the protocol. Do not let thaw before adding TRIzolTM. You may take out samples in batches of 4-6 samples.
SECTION: Isolate Snap Frozen Tissue or Cell P
20. In the fume hood, add 200 μl chloroform into a new and separate 1.5 ml tube.
SECTION: Isolate Snap Frozen Tissue or Cell P
21. Add 1000 μl lysate with TRIzolTM into each chloroform-containing 1.5 ml tube. This is best done in the fume hood. 
 

SECTION: Isolate Snap Frozen Tissue or Cell P
22. Gently invert tubes for 15-30 secs. Mix thoroughly to ensure proper phase separation. 
 

SECTION: Isolate Snap Frozen Tissue or Cell P
23. Incubate at room temp for 2-3 mins.
SECTION: Isolate Snap Frozen Tissue or Cell P
24. Centrifuge in a cold room at 4°C and 12,000xg for 15 mins.
SECTION: Isolate Snap Frozen Tissue or Cell P
25. Transfer 400 μl upper phase into each new 1.5 ml tube. 

 


SECTION: Isolate Snap Frozen Tissue or Cell P
26. Column-Based Method: Add 400-600 μl 70% ethanol into each RNA-containing tube. 
SECTION: Isolate Snap Frozen Tissue or Cell P
27. Column-Free Method: Add 0.5 ml of isopropanol to the aqueous phase to precipitate RNA, per 1 ml of TRIzolTM Reagent used for lysis.
SECTION: Isolate Snap Frozen Tissue or Cell P
25.1. Layers: (1) colorless upper aqueous phase containing RNA; (2) white interphase containing DNA; (3) red phenol-chloroform lower organic phase containing protein and lipids. heat produced while the samples were being homogenized. 
 

 

SECTION: Isolate Snap Frozen Tissue or Cell P
26.1. Gently pipette up-and-down 4-5 times to mix. RNA and ethanol should be 1:1 ratio. 

 

 

 

SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
28. Transfer 500 µl of sample into a spin cartridge with a collection tube.
SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
35. Add 500 µl wash buffer II to the spin cartridge and new collection tube.
SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
34.1. Note: steps #5-6 (i.e., wash buffer I steps) differ if DNase is to be added.
i. Add 350 µl wash buffer I to the spin cartridge.
ii. Centrifuge at room temp and 12,000g for 20 secs.
iii. Discard flow-through AND collection tube. Insert spin cartridge into new
collection tube.
iv. Add 80 µl PureLink DNase mixture to the center of the spin cartridge.
v. Incubate at room temp for 15 mins.
vi. Repeat steps #7 i-iii once more. Proceed to step #8.
SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
34. Discard flow-through AND collection tube. Insert spin cartridge into new collection tube.
SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
29. Centrifuge at room temp and 12,000xg for 20 secs.
SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
33. Centrifuge at room temp and 12,000xg for 20 secs
SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
32. Add 700 µl wash buffer I to the spin cartridge.
SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
31. Repeat steps #1-3 until all samples are processed.
 

SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
30. Discard flow-through ONLY.
SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
36. Centrifuge at room temp and 12,000xg for 20 secs.
SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
40. Discard flow-through AND collection tube. Insert spin cartridge into new 1.5 ml tube.
SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
39. Centrifuge at room temp and 12,000xg for 1 min to dry the spin cartridge before elution.
SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
38.  Repeat steps #8-10 once more.
SECTION: Bind and Wash Snap Frozen Tissues or Cell Pellets (Column-Based Method)
37.  Discard flow-through ONLY.
SECTION: ELUTE SNAP FROZEN TISSUES (Column-Based Method)
41. Add 30 µl RNase-free water to the center of the spin cartridge.
SECTION: ELUTE SNAP FROZEN TISSUES (Column-Based Method)
44. Store at -80°C. Avoid leaving RNA at room temp – Keep on wet ice.
SECTION: ELUTE SNAP FROZEN TISSUES (Column-Based Method)
42. Incubate at room temp for 30 secs.
SECTION: ELUTE SNAP FROZEN TISSUES (Column-Based Method)
43. Centrifuge at room temp and 12,000xg for 2 mins. Transfer eluent to a new 1.5 ml tube if broken.
 

SECTION: Elute Cell Pellets (Column-Based Method)
45. Add 20 µl RNase-free water to the center of the spin cartridge.
SECTION: Elute Cell Pellets (Column-Based Method)
50. Incubate at room temp for 30 secs.
SECTION: Elute Cell Pellets (Column-Based Method)
49. Add the eluent (i.e., 20 µl RNase-free water from step #1) to the center of the spin cartridge.
SECTION: Elute Cell Pellets (Column-Based Method)
46. Incubate at room temp for 30 secs.
SECTION: Elute Cell Pellets (Column-Based Method)
48. Important: Insert spin cartridge into new 1.5 ml tube. Keep the eluent for step #5.
SECTION: Elute Cell Pellets (Column-Based Method)
47. Centrifuge at room temp and 12,000xg for 2 mins.
SECTION: Elute Cell Pellets (Column-Based Method)
55. Store at -80°C. Avoid leaving RNA at room temp – Keep on wet ice.
SECTION: Elute Cell Pellets (Column-Based Method)
54.  Centrifuge at room temp and 12,000xg for 2 mins. Transfer eluent to a new 1.5 ml tube if broken.
SECTION: Elute Cell Pellets (Column-Based Method)
53. Incubate at room temp for 30 secs.
SECTION: Elute Cell Pellets (Column-Based Method)
52. Add new 10 µl RNase-free water to the center of the spin cartridge.
SECTION: Elute Cell Pellets (Column-Based Method)
51.  Centrifuge at room temp and 12,000xg for 2 mins.
SECTION: Wash and Solubilize Cell Pellets (Column-Free Method)
56. Wash the RNA
SECTION: Wash and Solubilize Cell Pellets (Column-Free Method)
56.1. Resuspend the cell pellet in 1 ml of 75% ethanol per 1 ml of TRIzol‱ Reagent used for
lysis.
SECTION: Wash and Solubilize Cell Pellets (Column-Free Method)
56.4. Discard the supernatant with a micropipette.
SECTION: Wash and Solubilize Cell Pellets (Column-Free Method)
56.3. Centrifuge in a cold room at 4°C and 7,500xg for 5 mins.
 

SECTION: Wash and Solubilize Cell Pellets (Column-Free Method)
56.2.  Mix the sample briefly.
SECTION: Wash and Solubilize Cell Pellets (Column-Free Method)
56.5. Air dry the RNA pellet for ~ 30 minutes at room temp.

 

SECTION: Wash and Solubilize Cell Pellets (Column-Free Method)
57. Solubilize the RNA 
SECTION: Wash and Solubilize Cell Pellets (Column-Free Method)
57.1. Resuspend the RNA pellet in 20–50 μl RNase-free water by pipetting up-and-down.
SECTION: Wash and Solubilize Cell Pellets (Column-Free Method)
57.2. Incubate in a heat block set at 55–60°C for 10–15 minutes.
SECTION: Wash and Solubilize Cell Pellets (Column-Free Method)
57.3. Proceed to downstream applications, or store the RNA at –70°C.