The first step will be to use liquid nitrogen to grind the tissues. This step is necessary to disrupt tissues and maximize cell exposure to the RNA extraction buffer, thus maximizing yields.
Material and reagents :
Procedure :
Under the hood: Add 8 µl of ß-mercaptoethanol to the 80O µl of RA1 extraction buffer. Be careful not to put ß-mercaptoethanol on the side of the tubes, as it has a pungent, bad smell.
Add one tungsten bead to each 2 ml Eppendorfs.
Label the Eppendorfs (your initials + sample, both on the side of the Eppendorfs and on the lid).
Harvest whole plants and add them into the tubes with the beads. Proceed quickly to step 4. Important note : the number of plants to use is important, as the extraction works best when using 100 mg of fresh tissue. This information will be provided at the moment of the harvest, as it depends on the growth stage of seedlings.
Tightly close the tubes and quickly add them to liquid nitrogen. Samples will be stable in liquid nitrogen. Remember to wear gloves and goggles
Then, we will use a « Tissue lyser » (= a machine that uses metal beads in order to grind tissues into a fine powder; see https://tinyurl.com/ys4hutsp) in order to crush the samples and grind them to a powder. This step will be performed by one of the supervisors.
Immediately after grinding, add 353 µl of the RA1+ß-mercaptoethanol buffer (=lysis buffer). The buffer will likely freeze because of the bead. Let it thaw at room temperature and Vortex vigorously.
Filter the lysate by filtration through NucleoSpin® Filter (violet ring). Be careful : you should never touch the silica membrane with your pipette or you’ll destroy it:
Important note: Do not disturb the pellet of cell debris at the bottom of the collecting tube, which may be visible after centrifugation.