Lu Lab Docs

6. How to purify RNA from blood

I. QIAzol法提取血浆RNA

  1. Prepare plasma, melt in room temperature.

  2. Add >3 volumes QIAzol Lysis Reagent. Mix by vortexing or pipetting up and down.

  3. Place the tube containing the lysate on the benchtop at room temperature (15–25°C) for 5 min.

  4. Add 0.2 ml chloroform per 1 ml QIAzol Lysis Reagent pipetted in step 1. Securely cap the tube containing the homogenate, and shake it vigorously for 15 s.

  5. Place the tube containing the lysate on the benchtop at room temperature (15–25°C) for 2–3 min.

  6. Centrifuge for 15 min at 12,000 x g at 4°C.

  7. Transfer the upper aqueous phase to a new collection tube. Avoid transfer of any interphase material. Add >1 volumes of 100% isopropyl alcohol and mix thoroughly by pipetting up and down several times.

  8. Place the tube at -20°C for at least 1h or overnight.

  9. Centrifuge at 16,000 x g for 30 min at 4°C.

  10. Carefully aspirate and discard the supernatant.

  11. Add at least 1 ml of 75% ethanol and flip the tube several times. Centrifuge at 16,000 x g for 10 min at 4°C.

  12. Remove the supernatant completely, and briefly air-dry the RNA pellet.

  13. Redissolve the RNA in an appropriate volume of RNase-free water.

  14. Dissolved RNA solution used for the folloting operation, or store at -80°C for long-term storage, 4°C for temporary storage(in one day.

II. Norgen法提取血浆RNA

  1. In a 50 mL tube (provided by the user), add 0.2 mL of Slurry C2 and 1.8 mL Lysis Buffer A (after the addition of β-mercaptoethanol) to 1 mL plasma/serum sample. Mix well by vortexing for 15 seconds. Note 1: Slurry C2 contains resin and must be mixed well before every pipetting

  2. Incubate the mixture from Step 1 for 10 minutes at 60oC.

  3. After incubation add 3 mL of 96-100% Ethanol (provided by the user). Mix well by vortexing for 15 seconds.

  4. Centrifuge for 30 seconds at 1,000 RPM, then carefully decant the supernatant in order to ensure that the slurry pellet is not dislodged.

  5. To the slurry pellet add 0.3 mL Lysis Buffer A, and mix well by vortexing for 15 seconds.

  6. Incubate the mixture from Step 5 for 10 minutes at 60oC.

  7. After incubation add 0.3 mL 96-100% Ethanol (provided by the user). Mix well by vortexing for 15 seconds.

  8. Transfer 650 ul from the mixture from Step 7 into a Mini Filter Spin column. Centrifuge for 1 minute at 14,000 RPM. Discard the flowthrough and reassemble the spin column with its collection tube;

  9. Repeat step 8 until all the mixture from Step 7 has been transferred to the Mini Filter Spin column.

  10. Apply 400 μL of Wash Solution A to the column and centrifuge for 1 minute at 14,000 RPM. Discard the flowthrough and reassemble the spin column with its collection tube.

  11. Repeat step 10 two more times, for a total of three washes.

  12. Spin the column, empty, for 3 minutes at 14,000 RPM. Discard the collection tube.

  13. Transfer the spin column to a fresh 1.7 mL Elution tube. Apply 100 ul of Elution Solution A to the column and centrifuge for 2 minutes at 2,000 RPM, followed by 3 minute at 14,000 RPM.

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