Sanger Tree of Life RNA Extraction: Automated MagMax™ mirVana

Prepare the TURBO DNase solution as described below, and once made, store on wet ice:

A	B
Turbo DNase (stored in freezer)	2.5
MagMAX TURBO DNase Buffer	60

Prepare the Binding Beads Mix as described below, and once made, store on wet ice:

A	B
RNA binding beads	12
Lysis/binding enhancer (stored in freezer)	12

Calculate the amount of lysis buffer required for the samples – 40 µL is required per 1 mg of tissue. 15 mg of sample is used for this protocol, so 600 µL per sample is required.

Create sufficient lysis buffer for your samples:

A	B
Lysis Buffer	1000
DTT	0.7

For samples that require powermashing, transfer 15 mg of tissue into a 1.5 mL BioMasher II tube and add 600 µL of lysis buffer. Disrupt sample in the lysis buffer using a PowerMasher II tissue disruptor and the BioMasher pestle, until no large pieces remain or sample cannot be disrupted further (for more detailed instructions regarding powermashing, please refer to the Sanger Tree of Life Sample Homogenisation: Powermash protocol).

For samples that have been cryoprepped, transfer the 15 mg of cryoprepped tissue into a 2 mL microcentrifuge tube and add 600 µL of lysis buffer. Pipette mix to homogenise the cryoprepped tissue and lysis buffer.

Incubate at room temperature for 30 seconds to 1 minute to allow samples to lyse. If samples will not immediately progress to Step 8, place samples on ice until ready to proceed.

Transfer 200 µL of each sample directly into individual wells of a Thermo Fisher KingFisher™ 1 mL 96-well deep-well plate.

Add 100 µL of isopropanol to each sample, seal the plate and shake at room temperature on a plate shaker for 2 minutes at 950 rpm.

Add 20 µL of the prepared binding beads mix to each sample, re-seal the plate and mix at room temperature on a plate shaker for 5 minutes at 950 rpm.

Prepare the remaining processing plates for the KingFisher™ Apex protocol:

A	B	C	D	E
Sample Plate	1	Deep-well	Sample + isopropanol + binding beads mix	200 µL sample + 100 µL isopropanol + 20 µL binding beads mix
Wash Plate 1	2	Deep-well	Wash solution 1	150 µL
Wash Plate 2	3	Deep-well	Wash solution 1	150 µL
DNase Plate	4	Deep-well	TURBO DNase solution	50 µL
Wash Plate 3	5	Deep-well	Wash solution 2	150 µL
Wash Plate 4	6	Deep-well	Wash solution 2	150 µL
Elution plate	7	Standard (200 µL)	Elution buffer	50 µL
Tip Comb	8	Deep-well	Place a tip comb in the plate

On the KingFisher™ Apex, select the protocol (details below in the KingFisher™ Apex RNA Extraction Protocol Script/attached KFX file in the Materials Section) on the protocols list and select using the play button.

Load the processing plates and the sample plate in the positions prompted by the instrument and then start the run. The full protocol will take approximately 50 minutes.

After 30 minutes, the protocol will pause and there will be a prompt to remove the DNase plate from the instrument, and add 50 µL of the rebinding buffer and 100 µL absolute isopropanol to each well containing sample as quickly as possible – do not premix these reagents and always add them separately to the wells.

Load the DNAse plate back into the instrument and press run to resume the protocol.

At the end of the run, remove the elution plate and store on ice.

Inspect the elution plates for any magnetic beads in the wells. In the rare instance of magnetic beads remaining in the eluate (possible in viscous samples), these samples will need to be transferred to a 1.5 mL microcentrifuge tube and placed on a magnetic rack. Allow around 5 minutes for the beads to migrate and take the clear eluate containing the RNA using a pipette tip.

Pipette the eluates into microcentrifuge tubes, perform the required QC, and then store eluates at –80°C.