TITLE: RNA Extraction from Frozen Brain Tissue Using a Bullet Blender
AUTHORS: Ashley V Kumar, Francesca Telese

[DESCRIPTION]
This protocol details a streamlined method for extracting high-quality RNA from small tissue samples using a bullet blender. By following this approach, researchers can efficiently isolate RNA suitable for a variety of downstream applications.

[BEFORE_START]
Bring centrifuges down to 4°C and follow guidelines for Workspace and Equipment Decontamination

[GUIDELINES]
Temperature Control: To maintain RNA integrity, keep all steps on ice whenever possible, unless otherwise specified in the protocol.
RNase Management for RNase-Rich Tissues: For tissues high in RNases (e.g., spleen, pancreas), pre-chill TRIzol‱ Reagent and other solutions to minimize RNA degradation.
Disposable, RNase-Free Equipment: Use only disposable, RNase-free pipette tips and tubes at each step to prevent contamination.
Glove Protocol: Always wear disposable gloves when handling RNA samples and reagents. Change gloves frequently—especially when transitioning from handling crude extracts to more purified materials—to minimize RNase transfer.
Workspace and Equipment Decontamination: Before starting, clean work surfaces and non-disposable items, such as centrifuges and pipettes, with RNaseZap‱ RNase Decontamination Solution (Cat. no. AM9780) to ensure an RNase-free environment.

[STEPS]
SECTION: Tissue homogenization and RNA extraction
1. Begin by resuspending the tissue sample in 500 µL of chilled TRIzol‱ Reagent. Transfer the mixture to a Safe-Lock tube, ensuring that the sample remains cold throughout handling. Add 10 µL of RNase-free Zirconium Beads (0.5 mm; Cat. no. ZrOB05-RNA) to the tube. Perform all steps on ice.
SECTION: Tissue homogenization and RNA extraction
2. Place the prepared Safe-Lock tubes in the Bullet Blender and process at speed 6 for 1 minute in a cold room. To optimize RNA quality, keep blending time as short as possible. Inspect samples after blending—if any tissue remains unhomogenized, repeat the processing step only for those specific samples, taking care not to over-homogenize.
SECTION: Tissue homogenization and RNA extraction
3. Briefly centrifuge the tubes at 1,000 rpm for 30 seconds at 4°C to bring any remaining TRIzol‱ Reagent down from the tube walls and lid for optimal RNA extraction. Carefully transfer the supernatant to a new RNase-free tube kept on ice
SECTION: Tissue homogenization and RNA extraction
4. Add 500 µL of chilled TRIzol‱ Reagent to the new tube containing the supernatant, bringing the final volume to 1 mL. Mix gently to ensure the sample is fully suspended in TRIzol‱.
SECTION: Tissue homogenization and RNA extraction
5. Working under the fume hood, add 0.2 mL of chloroform per 1 mL of TRIzol‱ Reagent used for lysis. Immediately cap the tube securely to prevent evaporation. If using TRIzol‱ LS, add 0.2 mL of TET buffer (0.05% Tween, 1 mM EDTA, 10 mM Tris, pH 7.5) instead of chloroform. 
SECTION: Tissue homogenization and RNA extraction
6. Incubate for 2–3 minutes at room temperature to facilitate phase separation.
SECTION: Tissue homogenization and RNA extraction
7. Briefly vortex the tube for 3–5 seconds, just until the mixture appears homogeneous. Avoid over-vortexing, as this may impact RNA integrity.
SECTION: Tissue homogenization and RNA extraction
8. Centrifuge the sample at 12,000 × g for 10 minutes at 4°C using a swinging bucket rotor. This centrifugation step will result in the separation of the mixture into three layers: a lower red phenol-chloroform phase (proteins and lipids), an interphase (DNA), and an upper colorless aqueous phase (RNA).
SECTION: Tissue homogenization and RNA extraction
9. While the centrifugation is in progress, add 1.5 µL of RNase-free GlycoBlue‱ Coprecipitant to a new, properly labeled tube. Note that GlycoBlue‱ co-precipitates with the RNA, aiding in visualization without interfering with downstream applications.
SECTION: Tissue homogenization and RNA extraction
10. Working under the fume hood, carefully transfer the upper, RNA-containing aqueous phase to the prepared tube (with GlycoBlue‱) at room temperature. Tilt the tube at a 45° angle and use a pipette to gently draw out the aqueous layer, being careful not to disturb or transfer any of the underlying TRIzol‱ or interphase. If there is any risk of contamination from the lower layers, perform a quick re-spin to ensure complete separation before attempting the transfer again.
SECTION: Precipitation, Washing and Elution
11. On the bench, add 1/10th volume of 3M sodium acetate (NaOAc) to the RNA-containing aqueous phase. Mix thoroughly by briefly vortexing, then spin down the tube to collect the contents at the bottom.
SECTION: Precipitation, Washing and Elution
12. Next, add 1 volume of 100% isopropanol to the aqueous phase mixture.  Then, vortex well to ensure thorough mixing
SECTION: Precipitation, Washing and Elution
13. Allow the RNA to precipitate by incubating the mixture for 20 minutes on ice, or alternatively, overnight at -20°C for maximum yield. Note: Avoid precipitation at -80°C, as any residual TRIzol‱ may also precipitate at this temperature, potentially contaminating the RNA.
SECTION: Precipitation, Washing and Elution
14. Centrifuge the tube at 28,000 × g (maximum speed) for 30 minutes at 4°C using a fixed-angle rotor. This step will pellet the RNA at the bottom of the tube for subsequent washes.
SECTION: Precipitation, Washing and Elution
15. Discard the supernatant with a micropipette, taking care not to disturb the RNA pellet. Quick spin and remove any leftover liquid with a micropipette.
SECTION: Precipitation, Washing and Elution
16. Add 1 mL of 75% ethanol per 1 mL of TRIzol‱ Reagent. Gently invert the tube to ensure the ethanol fully contacts the pellet and moves it around the tube. If needed, the RNA can be stored in 75% ethanol up to 1 year at -20°C or at least 1 week at 4°C.
SECTION: Precipitation, Washing and Elution
17. Centrifuge the tube at 500 × g for 1–2 minutes at 4°C (a quick spin is also acceptable) to bring the RNA pellet to the bottom. remove and discard the supernatant using a micropipette. Quick spin, then use a P20 pipette to carefully remove any remaining ethanol at room temperature.
SECTION: Precipitation, Washing and Elution
18. Let the RNA pellet air dry at room temperature for 5 minutes, or until it loses its shiny appearance.
SECTION: Precipitation, Washing and Elution
19. Add 12 µL of RNase-free water or TET to the pellet, adjusting the volume as needed for your desired concentration. Incubate at 55–60°C for 2 minutes, then immediately place on ice. Proceed to downstream applications or store at –80°C.