Purifying RNA from Animal and Plant Cells

Introduction This section provides instructions for purifying total RNA

from animal and plant cells. Separate protocols are provided
for ≤5 × 106 cells (suspension and monolayer) and for 5 × 106–
5 × 107 cells.

Materials You will need the following items in addition to the kit
Needed components:
• 2–mercaptoethanol
• 70% ethanol (in RNase-Free Water)
• Microcentrifuge capable of centrifuging 12,000 × g
• 1.5 mL RNase-free microcentrifuge tubes
• 15 mL RNase-free tubes (>107 cells per sample)
• PBS (>107 cells per sample)
• RNase-free pipette tips
• Optional: PureLink® DNase (page 67)
For ≤5 × 106 cells:
• Homogenizer (see page 67 and page 7) or,
• RNase-free syringe (1 mL) with 18-21 gauge needle or,
• Rotor-stator homogenizer (page 8)
For 5 × 106–5 × 107 cells:
Rotor-stator homogenizer (page 8)

Amount of Before beginning the lysis and homogenization steps,

Lysis Buffer prepare a fresh amount of Lysis Buffer containing 1%
Needed 2-mercaptoethanol for each purification procedure. Add
10 μL 2–mercaptoethanol for each 1 mL Lysis Buffer.
Using the table below, determine the correct amount of Lysis
Buffer needed for your sample type and amount.
Note: For larger than average samples, or if using a rotor-stator,
additional Lysis Buffer may be required. See page 12 for details.
Number of cells Amount of Lysis Buffer Needed
in your sample (prepared with 2-mercaptoethanol)
≤1 × 106 0.3 mL*
1 × 106–5 × 106 0.6 mL
5 × 106–5 × 107 0.6 mL per 5 × 106 cells
For example: use 1.2 mL for 1 × 107
cells and 6.0 mL for 5 × 107 cells
*Use 0.6 mL if using rotor-stator for lysis or homogenization.
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Purifying RNA from Animal and Plant Cells,
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For samples that are difficult to lyse, you can use TRIzol®
Reagent followed by purification using the PureLink® RNA
Mini Kit, (see page 49).

Lysis and Follow the steps below to prepare lysates from ≤5 × 106
Homogenization: suspension cells:
6
≤5 × 10 1. Transfer cells to an appropriately sized RNase-free tube
Suspension and centrifuge at 2,000 × g for 5 minutes at 4°C to pellet.
Cells Discard the growth medium from the tube.
2. Using RNase-free pipette tips, add the appropriate
volume of Lysis Buffer prepared with
2-mercaptoethanol to your sample (refer to previous
page for correct amounts).
3. Vortex at high speed until the cell pellet is completely
dispersed and the cells appear lysed.
Note: If you are using a rotor-stator, you may skip this step.
4. Proceed with one of the following homogenization
options at room temperature:
• Transfer the lysate to a Homogenizer (page 67)
inserted in a Collection Tube and centrifuge at
12,000 × g for 2 minutes. Remove the Homogenizer
when done, or
• Pass the lysate 5–10 times through an 18–21-gauge
needle attached to an RNase-free syringe, or
• Transfer the lysate to an appropriately sized
RNase-free tube and homogenize using a rotor-
stator homogenizer at maximum speed for at least
45 seconds. Centrifuge the homogenate at
~2,600 × g for 5 minutes, then transfer the
supernatant to a clean RNase-free tube.
Proceed to Binding, Washing, and Elution, page 19.

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Purifying RNA from Animal and Plant Cells,
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Lysis and Follow the steps below to prepare lysates from ≤5 × 106
Homogenization: monolayer cells:
6
≤5 × 10 1. Remove the growth medium from the cells.
Monolayer Cells
2. Using RNase-free pipette tips, add the appropriate
volume of Lysis Buffer prepared with
2-mercaptoethanol to your sample (refer to page 14 for
correct amounts).
3. Proceed with one of the following homogenization
options at room temperature:
• Transfer the lysate to a Homogenizer (page 67)
inserted in a Collection Tube and centrifuge at
12,000 × g for 2 minutes. Remove the Homogenizer
when done, or
• Transfer the lysate to a 1.5 mL RNase–free tube and
pass 5–10 times through an 18-21-gauge needle
attached to an RNase-free syringe, or
• Transfer the lysate to an appropriately sized
RNase-free tube and homogenize using a rotor-
stator homogenizer at maximum speed for at least
45 seconds. Centrifuge the homogenate at
~2,600 × g for 5 minutes, then transfer the
supernatant to a clean RNase-free tube.
Proceed to Binding, Washing, and Elution, page 19.

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Purifying RNA from Animal and Plant Cells,
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Lysis and Follow the steps below to prepare lysates from 5 × 106-–
Homogenization: 5 × 107 cells.
6 7
5 × 10 –5 × 10 Note: This protocol uses a rotor-stator homogenizer. Use 15 mL
Suspension RNase-free tubes to compensate for volume expansion that occurs
Cells during homogenization using a rotor-stator.
1. Transfer cells to a 15 mL RNase–free tube and
centrifuge at 2,000 × g for 5 minutes at 4°C to pellet.
Remove and discard the supernatant.
2. Using RNase-free pipette tips, add the appropriate
volume of Lysis Buffer prepared with
2-mercaptoethanol to your sample (refer to page 14 for
correct amounts).
3. Vortex at high speed until the cell pellet is completely
dispersed and the cells appear lysed.
4. Homogenize cells using a rotor-stator homogenizer at
maximum speed for at least 45 seconds.
5. Centrifuge the homogenate at ~2,600 × g for 5 minutes
at room temperature.
6. Transfer the supernatant to a clean 15-mL RNase–free
tube.
Proceed to Binding, Washing, and Elution (page 19).

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Purifying RNA from Animal and Plant Cells,
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Lysis and Follow the steps below to prepare lysates from frozen cell
Homogenization: pellets. For 5 × 106–5 × 107 cells, we recommend
Frozen Cell homogenizing with a rotor-stator homogenizer.
Pellets 1. Transfer the frozen cell pellet to an appropriately sized
RNase-free tube.
2. Using RNase-free pipette tips, add the appropriate
volume of Lysis Buffer prepared with
2-mercaptoethanol to your sample (refer to page 14 for
correct amounts).
Note: If you are using a rotor-stator homogenizer, you may
skip ahead to Step 4.
3. Vortex at high speed until the cell pellet is completely
dispersed and the cells appear lysed.
4. Proceed with one of the following homogenization
options at room temperature:
• Transfer the lysate to a Homogenizer (page 67)
inserted in an RNase-free tube and centrifuge at
12,000 × g for 2 minutes. Remove the Homogenizer
cartridge when done, or
• Pass the lysate 5–10 times through an 18–21-gauge
needle attached to an RNase-free syringe, or
• Transfer the lysate to an appropriately sized
RNase-free tube and homogenize using a rotor-
stator homogenizer at maximum speed for at least
45 seconds. Centrifuge the homogenate at
~2,600 × g for 5 minutes, then transfer supernatant
to a clean RNase-free tube.
Proceed to Binding, Washing, and Elution, next page.

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Purifying RNA from Animal and Plant Cells,
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Binding, Follow the steps below to bind, wash, and elute the RNA
Washing, and from your sample:
Elution 1. Add one volume 70% ethanol to each volume of cell
homogenate (prepared as described in the sample-
specific protocols (pages 15–18).
Note: If part of the sample was lost during homogenization,
adjust the volume of ethanol accordingly.
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.
Note: If you are processing the maximum starting amount of
sample, you may centrifuge for up to 10 minutes to completely
pass the lysate through the Spin Cartridge.
5. Repeat Steps 3–4 until the entire sample is processed.
Optional: If DNA-free total RNA is required, proceed
to On-column PureLink® DNase Treatment Protocol
(page 63).
6. Add 700 µL Wash Buffer I to the Spin Cartridge.
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.
7. Add 500 μL Wash Buffer II with ethanol (page 11) to the
Spin Cartridge.
8. 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.

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Purifying RNA from Animal and Plant Cells,
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Binding, 9. Repeat Steps 7–8 once.

Washing, and
10. Centrifuge the Spin Cartridge at 12,000 × g for
Elution, 1-2 minutes to dry the membrane with attached the
continued RNA. Discard the Collection Tube and insert the Spin
Cartridge into a Recovery Tube.
11. Add 30 μL–3 × 100 μL RNase–Free Water to the center
of the Spin Cartridge (see Elution Parameters, page 13).
12. Incubate at room temperature for 1 minute.
13. 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 you are performing sequential elutions, collect all
elutes into the same tube (see page 13 for Elution Parameters).
14. Store your purified RNA or proceed to Analyzing RNA
Yield and Quality (page 53) or to DNase I Treatment
After RNA Purification (page 65).

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