RNA extraction from Cyanobacteria using the Precellys 24

Sample: Cyanobacteria (Nostoc punctiforme)

Protocol:

Add cyanobacteria pellet to the 2 ml tough microorganism lysing kit.
Add PGTX buffer*.

PGTX buffer formula: phenol (39.6 g), glycerol (6.9 mL), 8-hydroxyquinoline (0.1 g), EDTA (0.58 g), sodium acetate (0.8 g), guanidine thiocyanate (9.5 g), guanidine hydrochloride (4.6 g) and Triton X-100 (2 mL). Final pH should be approximately 4.2. The PGTX extraction mixture should form a monophasic solution at room temperature.

Run the precellys at 6500rpm for 2 x 20 seconds with a 10s break.
Centrifuge the homogenate to remove any residual solids

Results:

RNA yield and purity were assessed using a NanoDrop ND-1000, an automated gel electrophoresis system, and RT-PCR. The Precellys 24 provided improved RNA yields and RNA purity compared to the user's previous method (high-temperature 95°C cell disruption in PGTX buffer). Use of PGTX buffer improved RNA purity and decreased experimental cost compared to Trizol.

Cyanobacteria RNA quality and yield after extraction using a Precellys 24

Extracted RNA yield and purity using Precellys with PGTX (left), Precellys with Trizol (center), and high-temperature lysis with PGTX (right).

RNA electrophoresis gel from Cyanobacteria lysed using a Precellys

Gel image of RNA obtained from cyanobacteria N. punctiforme using the Precellys with PGTX ("PGTX beads"), the Precellys with Trizol ("Trizol beads") or high-temperature lysis with PGTX ("PGTX 95").