Table 1 Steps of RNA extraction method from Maqui berry

1. Cell lysis

Grind 1.5 g fresh tissue to fine powder with a mortar and pestle under liquid nitrogen

Add 750 μl of preheated extraction buffer (65 °C)

Incubate at 65 °C for 10 min and agitate with a vortex for a few seconds during the incubation time

Centrifuge at 10,000×g (RCF) for 10 min at 4 °C

Recover the supernatant and mix with an equal volume of chloroform:IAA

Mix and centrifuge at 15,700×g (RCF) for 10 min at 4 °C

2. RNA precipitation

Recover the supernatant and add 1/4 volume of 10 M LiCl. Mix gently

Precipitate the RNA at −80 °C overnight

Centrifuge at 18,000×g (RCF) for 20 min at 4 °C

Wash the pellet with 500 µL of 70 % ice-cold ethanol

For ripe fruits, repeat this washing step at least twice, until the dark color disappears

Centrifuge briefly and remove the 70 % ethanol

Dissolve the RNA pellet in 100 µl of SSTE buffer. Briefly heat at 65 °C if required

3. RNA clean up

Add equal volume of phenol:chloroform:IAA (25:24:1) to the sample and shake vigorously

Incubate on ice for 5 min

Centrifuge at 18,000×g (RCF) for 20 min at 4 °C

Carefully transfer the supernatant to a new tube

Add equal volume of chloroform:IAA

Centrifuge at 18,000×g (RCF) for 20 min at 4 °C

Transfer the upper aqueous phase to a new tube

4. RNA precipitation

Precipitate RNA by adding an equal volume of absolute ethanol

Incubate at −20 °C for 2 h or at −80 °C for 30 min

Centrifuge at 18,000×g (RCF) for 15 min at 4 °C

Discard the supernatant

Carefully wash the pellet twice with chilled 70 % ethanol

Dry the pellet and dissolve in 200 μl DEPC-treated water