TITLE: RNA extraction using the PureLink® RNA Mini Kit
AUTHORS: Enrico Bagnoli, Miratul Muqit

[DESCRIPTION]
This Protocol details the extraction of total RNA using the PureLink® RNA Mini Kit.

[STEPS]
SECTION: Prepare lysates from ≤5 × 106 monolayer cells:
1. Remove the growth medium from the cells.


 

SECTION: Prepare lysates from ≤5 × 106 monolayer cells:
2. Using RNase-free pipette tips, add the appropriate volume of lysis buffer prepared with 2-mercaptoethanol to sample.

 

SECTION: Prepare lysates from ≤5 × 106 monolayer cells:
3. Proceed with one of the following homogenization options atRoom temperature:

SECTION: Prepare lysates from ≤5 × 106 monolayer cells:
3.1. Transfer the lysate to a homogenizer inserted in a collection tube and centrifuge at  12000 x g, 2 min. Remove the homogenizer when done, or
SECTION: Proceed to Binding, Washing, and Elution
4. 
 

Add one volume 70% ethanol to each volume of cell homogenate (prepare the sample as described in specific protocols.

 

SECTION: Proceed to Binding, Washing, and Elution
5. Vortex to mix thoroughly and to disperse any visible precipitate that may form after adding ethanol.
SECTION: Proceed to Binding, Washing, and Elution
6. Transfer up to 700 µLof the sample (including any remaining precipitate) to the Spin Cartridge (with the Collection Tube).

SECTION: Proceed to Binding, Washing, and Elution
7. Centrifuge at 12000 x g, 15 s . 

Discard the flow-through, and reinsert the Spin Cartridge into the same collection tube.

 

SECTION: Proceed to Binding, Washing, and Elution
8. Repeat Steps 6–7 until the entire sample is processed. 

 

SECTION: Proceed to Binding, Washing, and Elution
9. Add 700 µL wash buffer I to the Spin Cartridge. 

Centrifuge at 12000 x g, 15 s. Discard the flow-through and the Collection Tube. 
Place the Spin Cartridge into a new Collection Tube.

SECTION: Proceed to Binding, Washing, and Elution
10. Add 500 µLWash Buffer II with ethanol to the Spin Cartridge.

SECTION: Proceed to Binding, Washing, and Elution
11. Centrifuge at 12000 x g, 15 s.

Discard the flow-through and reinsert the Spin Cartridge into the same collection tube.

SECTION: Proceed to Binding, Washing, and Elution
12. Repeat Steps 10–11 once.
SECTION: Proceed to Binding, Washing, and Elution
13. Centrifuge the Spin Cartridge at 12000 x g for 1 min-2 min to dry the membrane with attached the RNA. Discard the collection tube and insert the Spin Cartridge into a recovery tube.

SECTION: Proceed to Binding, Washing, and Elution
14. Add 30 µL–3 × 100 µL RNase–Free Water to the center of the Spin Cartridge (see Elution Parameters).


 

SECTION: Proceed to Binding, Washing, and Elution
15. Incubate at Room temperature for 1 min.

SECTION: Proceed to Binding, Washing, and Elution
16. Centrifuge the Spin Cartridge for 2 min at ≥ 12000 x g to elute the RNA from the membrane into the recovery tube.

 

SECTION: Proceed to Binding, Washing, and Elution
17. Store your purified RNA or proceed to Analyzing RNA Yield and Quality or to DNase I Treatment after RNA purification.
SECTION: Prepare lysates from ≤5 × 106 monolayer cells:
3.3. Transfer the lysate to an appropriately sized RNase-free tube and homogenize using a rotorstator homogenizer at maximum speed for at least 45 s. 

Centrifuge the homogenate at ∼2600 x g, 5 min, then transfer the supernatant to a clean RNase-free tube.

SECTION: Prepare lysates from ≤5 × 106 monolayer cells:
3.2. 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
Centrifuge the homogenate at ∼2600 x g, 5 min, then transfer the supernatant to a clean RNase-free tube