RNA extraction from mycotoxigenic filamentous fungi

Instrument: Precellys® 24 Dual vs Mortar and pestle.

Sample: Mycelium of A. flavus NRRL 3357

Protocol:

Precellys kits: CK14, CK28, VK01, VK05 (2mL vials).
Sample: ~150 mg of mycelium of A. flavus NRRL 3357.
Lysis buffer: 750 μL, either TRIzol or RLT buffer (provided with Qiagen RNeasy plant mini kit) supplemented with β-mercaptoethanol.
Run the Precellys 24 at 6500 rpm, 2x25 secs ( 5 sec break).
The lysate was centrifuged at +4ºC for 5-10 minutes at 16000 g for an initial homogenization.
RNA purification: Qiagen RNeasy plant mini kit, QIAcube® robotic workstation.

Results:

Significant differences were found between the total RNA amounts isolated using the Precellys 24 homogenizer and the manual system (p-value=0.0072).

Furthermore the use of glass beads resulted in a yield around 3 times higher than using the traditional method of the mortar and pestle. The high quality of the RNA as also achieved as an example the electropherogram of a high quality RNA sample extracted using the 0.5 mm glass beads.

The use of the Precellys 24 with the prefilled Precellys lysing kit VK05 significantly improved the concentration and the quality of the total RNA extracted from mycotoxigenic filamentous fungi while reducing the required amount of mycelium.

Total RNA yield average per 100 mg of initial biomass and standard deviation comparing different beads with the manual method. Key to treatments: CK – Zirconnium Oxide, VK – Glass; followed by the bead size code 01 – 0.1 mm, 05 – 0.5 mm, 14 – 1.4 mm, 28 – 2.8 mm.

Electropherogram of a good quality RNA sample extracted using Precellys at room temperature. The RQI of this sample is 9.7.