Programa de capacitación y sensibilización sobre residuos sólidos

DsRNA was extracted from fungal tissues using different methods depending upon the quantity and need.

2.7.7.1 Extraction of total nucleic acid (LiCl fractionation).

This procedure is adapted from Hull and Covey (1983) and is schematically represented in Fig. 2.2. Fungal mycelia were harvested and powdered in liquid nitrogen. Extraction buffer (50 ml/l Tris-HCl buffered phenol [Sigma], 60 g/l 4-aminosalicylate [Sigma], made up to the

desired volume with autoclaved 50 mM Tris-HCl, [pH 8.4]) was added to the powder and the mixture shaken on an ice plate for 60 min. The mixture was centrifuged (15,300 x g; 40 min). The aqueous supernatant containing total nucleic acids was transferred into a sterile centrifuge tube and phenol: chloroform extracted (section 2.7.6.1) to deproteinise the nucleic acids. The mixture was centrifuged (15,300 x g; 30 min) on each occasion. After extraction, LiCl (Sigma Aldrich) fractionation was carried out according to Diaz-Ruiz and Kaper (1978). The supernatant was transferred to a sterile tube and an equal amount of 4 M LiCl was added, mixed and then incubated overnight at 4oC. The solution was then centrifuged (15,300 x g; 30 min) to pellet the precipitated ssRNA. The supernatant was transferred to a sterile tube and an equal volume of 8 M LiCl added, mixed and incubated overnight at 4oC. The solution was centrifuged at (15,300 x g; 45 min) to collect the dsRNA. A sample (1ml) was taken from solution before discarding it carefully leaving the pellet. This sample was used as negative control (eluate). The pellet was then resuspended in water. The dsRNA was then precipitated from the aqueous phase as described in section 2.7.6.2 and either stored as a precipitate at -70oC or dried, and resuspended in water for further manipulation.

Homogenised mycelia + extraction buffer 15,300 x g; 30 min Supernatant + phenol 15,300 x g; 30 min Supernatant + chloroform 15,300 x g; 30 min

Supernatant + LiCl (4M) precipitation

15,300 x g; 30 min

Supernatant + LiCl (8M) precipitation overnight

15,300 x g; 45 min

dsRNA ethanol precipitation at -20°C

DNase/S1 nuclease treatment

Phenol/Sevag treatment

Ethanol precipitation overnight at -20°C

1% agarose gel Negative control (eluate)

2.7.7.2 Virus purification

The virus from A. fumigatus isolate A-56 was purified using differential centrifugation (Kim and Bozarth, 1985). Harvested mycelia was removed from -80oC storage and allowed to thaw. Then 10g of silica carbide C6 was added per 25 g of mycelia. Mycelia were then homogenised with silica carbide (abrasive grade) in a pestle and mortar using 20 ml/25 g of cold PBK buffer (0.1 M disodium hydrogen phosphate (Na2HPO4.2H2O), 0.1 M sodium

dihydrogen orthophosphate 1-hydrate (NaH2PO4.H2O), 0.1 M potassium chloride (KCl)/l, pH

[7.0]) to resuspend the homogenate. The mixture was then stirred at 4oC for 4 h. Cell debris was then removed by centrifugation (12,000 rpm; 45 min) in a Beckman JA-14 rotor at 4oC. The supernatant was transferred into a beaker and polyethylene glycol (PEG; Sigma) and NaCl (Melford) was added to achieve 10 % (w/v) and 0.15 M concentrations respectively. The solution was stirred at 4oC for 2 h to precipitate virions. Precipitated virions were pelleted by centrifugation (12,000 rpm; 45 min) in a Beckman JA-20 rotor at 4oC. The supernatant was discarded and the pellets were resuspended in a small amount of cold PBK buffer. Virus particles were pelleted by ultracentrifugation (30,000 rpm; 3.5 h) in a Beckman Type 35 rotor at 4oC. The pellet was resuspended in 1 ml of cold PBK buffer and layered on top of a continuous sucrose density gradient (10 %-50 %, prepared in PBK buffer). Ultracentrifugation (33,700 rpm; 3 h) in a Beckman SW 41Ti rotor at 4oC was then performed to band the virus particles according to its relative density.

2.7.7.3 Extraction of viral dsRNA using cellulose CF-11

The protocol was adapted from Coffin and Coutts (1992) for the extraction of dsRNA from plant tissues. Freshly harvested 100 gm of mycelia was homogenised in liquid nitrogen and added into a mixture containing 150 ml GPS (GPS buffer 5x: 87.66 g NaCl, 37.54 g glycine, 35.49 g Na2HPO4, [pH 9.5]), 150 ml GPS-saturated phenol, 150 ml Sevag, 15 ml 10 % SDS

and 1.5 ml β-mercaptoethanol and was left to stir at 4o_{C for 1 h. The material was strained}

through a double layered muslin cloth to remove solid material and subjected to centrifugation (13,000 x g; 10 min) at 4oC. The aqueous phase was removed and ethanol was added to a final concentration of 17% together with CF-11 cellulose (3 g/l) and the mixture stirred for 1 h to allow binding of dsRNA and then centrifuged (13,000 x g; 10 min). The resulting pellet was resuspended in ca. 45 ml of STE buffer (STE buffer 10x: 29.22 g NaCl, 30.29 g Tris-HCl, 1.86 g EDTA, 500 ml H2O, [pH 7.5] ) with 17% ethanol and shaken for 5 min and the cellulose was repelleted by centrifugation (2000 x g; 2 min). This step was repeated four times. The dsRNA was eluted using 20 ml of STE buffer (without ethanol) in four steps and centrifuged as before, with the final elution being left for 1 h on a rotating wheel to maximise yield. The combined supernatants were collected and centrifuged (23,700 x g; 30 min) to remove any residual cellulose. Nucleic acids were then precipitated from the

supernatant as described in section 2.7.6.2. Following overnight precipitation dsRNA was pelleted, dried and resuspended in 1 ml aliquots and frozen at -80oC prior to be being treated with DNase and S1 nuclease (Section 2.7.6.5) and agarose gel electrophoresis analysis.

2.7.7.4 Extraction of total RNA (RNeasy Plant Mini Kit)

For the extraction of total RNA from mycelium the RNeasy plant minikit (QIAGEN) was used. Approximately 100 mg of mycelia was crushed under liquid nitrogen and immediately put into an Eppendorf tube. An appropriate amount of buffer RLC (450 l) containing β- mercaptoethanol (320 mM) was added and vortexed vigorously for 30 sec followed by incubation at 56oC for 3 min. The lysate was transferred to a QIAshredder spin column placed in a 2 ml collection tube and centrifuged (13,000 x g; 2 min). The supernatant was carefully pipetted into a new Eppendorf tube without disturbing the pellet and 0.5 vols of ethanol was added to the cleared lysate and mixed immediately by pipetting, followed by transfer of the sample to an RNeasy spin column placed in a 2 ml collection tube and centrifuged (13,000 x g; 15 sec). The flow through was discarded and 650 l of buffer RW1 was added to the column, gently closing the lid and centrifuged again (13,000 x g; 15 sec) followed by addition of 450 l of buffer RPE to the column after discarding the flow through and re-centrifugation (13,000 x g; 15 sec). The step was repeated after discarding the flow through and the column was placed in a new collection tube andcentrifuged (13,000 x g; 1 min) to remove any residual RPE buffer. The column was placed in a new 1.5 ml collection tube and 30 l of sterile water was added and centrifuged (13,000 x g; 1 min) to elute the RNA.

2.7.7.5 Extraction of dsRNA (small scale extraction).

The protocol used was adapted from Coenen et al. (1997) and was used to identify the presence of dsRNA in small amounts of mycelia. Approxiately 500-1000 mg of mycelia was homogenised in liquid nitrogen and 350 l of extraction buffer (20 mM EDTA, 20 mM Tris- HCl [pH 7.5], 1% SDS and 1% NaCl) added and the solution was vortexed vigorously for 30 sec in a 2 ml Eppendorf tube. The mixture was incubated at 70oC for 1 h and an equal amount of phenol and Sevag added, vortexed and centrifuged (11,000 x g; 15 min) following incubation. After centrifugation, 500 l of Sevag was added to the supernatant, vortexed and re-centrifuged (11,000 x g; 10 min). The resulting nucleic acid was precipitated with 2.5 vols of 100% ethanol for 4 h. Samples were subjected to centrifugation (11,000 x g; 10 min) and after discarding the supernatant pellets were dried for 15-20 min and resuspended in appropriate amounts of waterand analysed on 1% agarose gels.