Modified from the E.Z.N.A.^® Plant RNA Kit, these kits use an improved buffer system that is more efficient for fungal samples. The E.Z.N.A.^® Fungal RNA Kits provide convenient and rapid methods for the isolation of total RNA from a variety of fungal species. These kits do not require the use of cumbersome or expensive shredding/homogenizing accessories in an attempt to shear genomic DNA in viscous cell lysates. In combination with HiBind^® RNA spin-columns, this procedure permits purification of high quality RNA from as much as 100 mg tissue. The system is efficient enough to allow isolation of total RNA from as little as 10 mg of tissue or 100 cells. E.Z.N.A.^® Fungal RNA Kits are ideal for processing multiple samples in less than one hour. Kits can also be used to purify RNA from enzymatic reaction mixtures and to desalt RNA. Purified RNA has an OD ratio of 1.8-2.0.

Principle

E.Z.N.A.^® Fungal RNA Kit involves a simple and rapid precipitation step for removal of polysaccharides and phenolic compounds commonly found in fungal tissues. The E.Z.N.A^® Fungal RNA Kits use reversible binding properties of HiBind^® matrix, a new silica-based, time saving spin technology material. HiBind™ matrix avidly, but reversibly, binds RNA under certain optimal conditions allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with de-ionized water or low salt buffer.

Procedure

Collect frozen ground fungal sample (up to 100 mg) in a microfuge tube and immediately add 500 µl Buffer RB/2-mercaptoethanol. Transfer the lysate into a Homogenization Spin Column and centrifuge at 13,000 x g for 5 min at room temperature. Collect the supernatant in a new microcentrifuge tube and add 0.5 volume absolute ethanol. Apply the entire sample to a HiBind^® RNA spin column. With a brief centrifugation or vacuum, the samples pass through the column and the RNA binds to the Hibind^® matrix. Trace DNA that may have co-purified with the RNA can be removed by an on column DNase treatment. After three wash steps (Once with RNA wash buffer I and twice with RNA wash buffer II diluted with ethanol), purified total RNA is eluted with RNase-free DEPC-treated water.

Applications

RNA purified using the E.Z.N.A.^® Fungal RNA Kit can be used in applications such as :

• RT-PCR
• Northern Analysis
• Differential display
• Poly A⁺ RNA selection