96–100% ethanol
2–mercaptoethanol
70% ethanol (in RNase-Free Water)
1.5 mL RNase-free microcentrifuge tubes
RNase-free pipet tips
Homogenizer, RNase-free syringe (1 mL) with 18–21-gauge needle
or, Rotor-stator homogenizer
Microcentrifuge capable of centrifuging 12,000 × g
15 mL RNase-free tubes (for samples with >107 cells)
1. Lysis and Homogenization
1) ≤5 × 106 Suspension Cells
i. Transfer the cells to an RNase-free tube and centrifuge at 2,000 × g for 5 min at 4°C to pellet. Discard the growth medium.
ii. Add 0.3 or 0.6 mL Lysis Buffer with 2-mercaptoethanol to the sample (see table above for volume).
iii. Vortex until the cell pellet is dispersed and the cells appear lysed.
iv. Proceed to Homogenization below.
2) ≤5 × 106 Monolayer Cells
i. Remove the growth medium from the cells, then add 0.3 or 0.6 mL. Lysis Buffer with 2-mercaptoethanol (see table above for volume).
ii. Vortex until the cell pellet is dispersed and the cells appear lysed.
iii. Proceed to Homogenization below.
3) 5 × 106 –5 × 107 Suspension Cells
i. Transfer cells to a 15-mL tube and centrifuge at 2,000 × g for 5 min at 4°C. Discard the supernatant.
ii. Add 0.6 mL Lysis Buffer with 2-mercaptoethanol (see table above for volume).
iii. Vortex until the cell pellet is dispersed and the cells appear lysed.
iv. Homogenize at room temperature with a rotor-stator homogenizer (see Homogenization below).
4) Frozen Cell Pellets
i. Transfer cells to a 15-mL tube and add 0.6 mL Lysis Buffer with 2-mercaptoethanol (see table above for volume).
ii. Vortex until the cell pellet is dispersed and the cells appear lysed.
iii. Homogenize at room temperature with a rotor-stator homogenizer (see Homogenization below).
5) Homogenization
i. Proceed with one of the following homogenization options at room temperature.
ii. Transfer the lysate into a clean homogenization tube, and perform manual homogenization. Centrifuge the homogenate at 12,000 × g for 2 minutes.
iii. Pass the lysate 5–10 times through an 18- to 21-gauge syringe needle.
iv. Transfer the lysate into a clean tube, and homogenize using a rotor-stator homogenizer at maximum speed for ≥45 s. Centrifuge the homogenate at 26,000 × g for 5 minutes, then transfer the supernatant to a clean RNase-free tube.
v. Proceed to RNA Purification
2. RNA Purification
Binding, Washing, and Elution of RNA
1) Add one volume 70% ethanol to each volume of cell homogenate.
2) Vortex to mix thoroughly and to disperse any visible precipitate that may form after adding ethanol.
3) Transfer up to 700 μL of the sample (including any remaining precipitate) to the spin cartridge (with the collection tube).
4) Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and reinsert the spin cartridge into the same collection tube.
5) Repeat Steps 3–4 until the entire sample has been processed.
6) Add 700 μL Wash Buffer I to the spin cartridge.
7) Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through and the collection tube. Place the spin cartridge into a new collection tube.
8) Add 500 μL Wash Buffer II with ethanol to the spin cartridge.
9) Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through.
10) Repeat Steps 8–9 once.
11) Centrifuge the spin cartridge at 12,000 × g for 1–2 minutes to dry the membrane with bound RNA. Discard the collection tube and insert the spin cartridge into a recovery tube.
12) Add 30–100 μL RNase–free water to the center of the spin cartridge.
13) Incubate at room temperature for 1 minute.
14) Centrifuge the spin cartridge for 2 minutes at ≥12,000 × g at room temperature to elute the RNA from the membrane into the recovery tube. Note: If the expected RNA yield is >100 μg, perform 3 sequential elutions of 100 μL each. Collect the eluates in a single tube.
15) Store your purified RNA or proceed to downstream application.
3. RNA Storage
Store the purified RNA on ice for immediate use. For long–term storage, keep the purified RNA at –80°C. Perform DNase I treatment after purification (refer to the PureLink™ RNA Mini Kit manual) to assure highly pure RNA without genomic DNA contamination. Determine the quality and quantity of your RNA by UV absorbance at 260 nm.
1. Keep freshly harvested samples on ice and quickly proceed to Lysis and Homogenization, or freeze samples immediately after collection in liquid nitrogen or on dry ice and keep at –80°C for later use.
2. Do not exceed the RNA binding capacity of the spin cartridge by adding samples containing more than 1 mg of total RNA.
3. Both Lysis Buffer and Wash Buffer I contain guanidine isothiocyanate. Do not add bleach or acidic solutions directly to solutions or sample preparation waste containing guanidinium isothiocyanate, as reactive compounds and toxic gases are generated.
4. Solutions containing ethanol are considered flammable. Use appropriate precautions when using this chemical.
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