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I hope everyone is ok. I'm looking for a protocol to isolate muscle RNA using zirconium beads.
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Isidora
Deokrishna Kumar Choudhary
Tissue Disruption:a. Weigh the muscle tissue sample and place it in a suitable tube. b. Add TRIzol Reagent to the tube at a ratio of about 1 mL TRIzol per 50-100 mg of tissue. c. Add zirconium beads to the tube. The bead size will depend on the homogenizer you are using. Follow the manufacturer's recommendations for the appropriate bead size and number. Usually, 2-3 zirconium beads are sufficient. d. Homogenize the tissue using a homogenizer. Typically, multiple 20-30 second cycles of homogenization with cooling on ice between cycles are recommended. Be sure to use a homogenization speed suitable for your tissue type. Phase Separation:a. After homogenization, incubate the sample at room temperature for 5 minutes to allow complete dissociation of nucleoprotein complexes. b. Add chloroform to the sample in a ratio of 0.2 mL chloroform per 1 mL of TRIzol used in step 1b. c. Shake vigorously for 15 seconds and incubate at room temperature for 2-3 minutes. d. Centrifuge the sample at 12,000 x g for 15 minutes at 4°C. After centrifugation, the mixture will separate into three phases: a lower red organic phase, an interphase, and a colorless upper aqueous phase containing RNA. RNA Precipitation:a. Carefully transfer the upper aqueous phase (containing RNA) to a new tube. b. Add an equal volume of isopropanol to the aqueous phase and mix gently by inverting the tube several times. c. Incubate the mixture at -20°C or -80°C for at least 30 minutes to precipitate the RNA. RNA Pellet Collection:a. Centrifuge the sample at 12,000 x g for 10-15 minutes at 4°C to pellet the RNA. b. Carefully remove and discard the supernatant without disturbing the RNA pellet. RNA Wash:a. Wash the RNA pellet with 75% ethanol by adding 1 mL of ice-cold 75% ethanol to the pellet. b. Centrifuge the sample at 7,500 x g for 5 minutes at 4°C. c. Carefully remove the ethanol without disturbing the RNA pellet. RNA Resuspension:a. Allow the RNA pellet to air-dry for 5-10 minutes or until the ethanol odor is completely gone. b. Add an appropriate volume of RNase-free water to resuspend the RNA pellet. The volume will depend on the size of your pellet and the desired RNA concentration. RNA Storage:a. Store the isolated RNA at -80°C or proceed with downstream applications such as reverse transcription or quantitative PCR.
Isolating RNA from muscle tissue using the zirconium bead method involves mechanical disruption of the tissue using zirconium beads and a homogenizer, followed by RNA extraction using a suitable RNA extraction kit. Here's a step-by-step protocol:
Materials Needed:
- Muscle tissue samples
- TRIzol Reagent or a similar RNA extraction reagent
- Zirconium beads (various sizes, depending on the homogenizer)
- Homogenizer (e.g., Bead Mill, TissueLyser)
- Chloroform
- Isopropanol
- 75% ethanol
- RNase-free tubes and pipette tips
- Microcentrifuge
- RNAse-free water
Protocol:
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