RNA extraction using the PureLink® RNA Mini Kit

Enrico Bagnoli, Miratul Muqit

Published: 2024-05-14 DOI: 10.17504/protocols.io.261ge54r7g47/v1

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Abstract

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

Steps

Prepare lysates from ≤5 × 106 monolayer cells:

Remove the growth medium from the cells.

Note

We used a 15 cm² dish for each sample. The cells, collected in 1 ml of DPBS, were then divided, with ⅔ used for western blotting, and ⅓ used for the following protocol for RNA extraction.

Using RNase-free pipette tips, add the appropriate volume of lysis buffer prepared with 2-mercaptoethanol to sample.

Proceed with one of the following homogenization options atRoom temperature:

3.1.

Transfer the lysate to a homogenizer inserted in a collection tube and centrifuge at 12000x g. Remove the homogenizer when done, or

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 ∼ , then transfer the supernatant to a clean RNase-free tube 2600x g, then transfer the supernatant to a clean RNase-free tube

Note

We homogenized the cells by 10 passages in a 20G needle in 300µL of lysis buffer

3.3.

Transfer the lysate to an appropriately sized RNase-free tube and homogenize using a rotorstator homogenizer at maximum speed for at least 0h 0m 45s.

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

Proceed to Binding, Washing, and Elution

Note

Follow the steps below to bind, wash, and elute the RNA from your sample:

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

Note

If part of the sample is lost during homogenization, adjust the volume of ethanol accordingly.

Vortex to mix thoroughly and to disperse any visible precipitate that may form after adding ethanol.

Transfer up to 700µLof the sample (including any remaining precipitate) to the Spin Cartridge (with the Collection Tube).

Centrifuge at 12000x g .

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 0h 10m 0s to completely pass the lysate through the Spin Cartridge.

Repeat Steps 6–7 until the entire sample is processed.

Note

Optional: If DNA-free total RNA is required, proceed to On-column PureLink® DNase Treatment Protocol.

Add 700µL wash buffer I to the Spin Cartridge.

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

10.

Add 500µLWash Buffer II with ethanol to the Spin Cartridge.

11.

Centrifuge at 12000x g.

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

12.

Repeat Steps 10–11 once.

13.

Centrifuge the Spin Cartridge at 12000x g,0h 0m 0s for 0h 1m 0s-0h 2m 0s to dry the membrane with attached the RNA. Discard the collection tube and insert the Spin Cartridge into a recovery tube.

14.

Add 30µL–3 × 100µL RNase–Free Water to the center of the Spin Cartridge (see Elution Parameters).

Note

We diluted in 50µL of water

15.

Incubate at Room temperature for 0h 1m 0s.

16.

Centrifuge the Spin Cartridge for 0h 2m 0s at ≥ 12000x g,0h 0m 0s 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 Elution Parameters).

17.

Store your purified RNA or proceed to Analyzing RNA Yield and Quality or to DNase I Treatment after RNA purification.