Methods of dsRNA extraction new 2024.pptx

Fungi

1. DsRNA Isolation Procedures from fungi

2. Educational Objective of Today’s lecture Introduce students to diverse ways of isolating dsRNA from fungi New and reported procedures Kit based protocols etc

3. Mentimeter time! 7726 0199

4. Total nucleic acid extraction Lithium chloride fractionation RNAeasy MiniKit Extraction using cellulose powder Via purified virus particles Non phenol/chloroform extraction

5. 1. Total Nucleic Acid Extraction* Grinding of mycelia in liquid nitrogen Addition of extraction buffer (200mM NaCl , 100 mM Tris-HCl , 4mM EDTA, 2% SDS) incubation at 70 o C for 60 mins Clearing with Phenol and Sevag ( Chl:IAA ) Addition of 500 microliters Sevag Precipitation of total nucleic acid with 2.5 vol of 100% ethanol+ 10 % Sodium acetate 3M Incubate at -20 o C for 4 hrs /overnight Centrifuge and discard EtOH . Dry pellet and suspend in sterile distilled water Analysis of total nucleic acid by gel electrophoresis * Coenen , A., Kevel, F. and Hoekstra, R. F., (1997). Gen. Res., 69, 1-10.

6. 2. Lithium Chloride Fractionation* Grinding of mycelia (25-30 g) with Liq. N2 Addition of extraction buffer Leave on for stirring for 60 mins clearing with Phenol clearing with Chloroform Supernatant mixed with 4M lithium chloride Overnight incubation at 4 o C *Diaz-Ruiz, J. and Kaper , J., (1978). Prep. Biochem ., 8, 1-17.

7. Cont … Centrifuge to pellet down ssRNA and dsDNA Supernatant mixed with 8M lithium chloride Overnight incubation at 4 o C Centrifuge to pellet down dsRNA Dry the pellet at room temperature Re-suspend pellet in sterile distilled water

8. RNAeasy Minikit

9. Extraction Using Cellulose Powder * (small scale) Homogenization of mycelia in liquid nitrogen (~1 g) addition of extraction buffer and phenol (1ml each) centrifuge at 4 o C Add 1ml Chl /IAA and centrifuge Add 16% vol. ethanol and cellulose powder (0.1g) to the aqueous phase *Eusebio-Cope, A., and Suzuki, N. (2015). Nucleic Acids Res. 43, pp. 3802–3813.

10. Cont. Leave for stirring for an hour to allow binding of dsRNA to cellulose Centrifuge to pellet down cellulose + dsRNA Wash pellet in STE buffer + EtOH (three cycles) Elution of dsRNA in STE buffer (followed by centrifugation) Precipitation of dsRNA with 3M NaOAC and 100% EtOH Incubation at 4 o C for 4 hrs / overnight OR at -80 o C for 30mins Centrifugation at top speed for 20 to 30 mins S Salt ( NaCl ) T Tris-HCl EEDTA

11. Cont.. Washing of pellet with 70% EtOH Dry the pellet and and resuspend in sterile distilled water Gel electrophoresis

12. Non Phenol Chloroform Method* Grinding of mycelia in liquid nitrogen Add 1ml EBA modified buffer and mix well Spin at 4 o C at 13k rpm for 10 mins Transfer supernatant (~700-750µl) to fresh tubes Add equal volume of a mixture of cellulose+ EtOH+H 2 O Add 30µl of 100 mM NaCl EBA Buffer: 50 mM tris HCl , 50 mM EDTA, 2% SDS , 1% PVPP, ( Polyvinylpolypyrrolidone ) 50mM DTT Dithiothreitol   * Ballija et al. (2008). Journal of Virological Methods 152 (2008) 32–37

13. Cont. Leave for stirring for an hour to allow binding of dsRNA to cellulose Centrifuge to pellet down cellulose + dsRNA Wash pellet in STE buffer + EtOH (three cycles) Elution of dsRNA in STE buffer (followed by centrifugation) Precipitation of dsRNA with 3M NaOAC and 100% EtOH Incubation at 4 o C for 4 hrs / overnight OR at -80 o C for 30mins Centrifugation at top speed for 20 to 30 mins S Salt ( NaCl ) T Tris-HCl EEDTA

14. Cont.. Washing of pellet with 70% EtOH Dry the pellet and and resuspend in sterile distilled water Gel electrophoresis

15. Via Purified Virus Particles Homogenization of mycelia with carborundum and phosphate buffer Raise volume to 1.5 litres and stir at 4 o C for 1 hr Centrifuge at 23,000 X g at 4˚C for 45 mins. Add PEG 6000 and NaCl to the supernatant Leave for stirring for 2hrs at 4˚C Centrifuge at 15,000 X g for 35 mins.

16. Cont … Resuspend pellet in PBK buffer and leave overnight at 4˚C low speed centrifugation at 23,000 X g for 30 min Ultracentrifugation at 80,110 X g for 4 h Suspend pellet in small quantity of PBK buffer Crude/ partially purified particles loaded over a sucrose density gradient (10-50% w/v)

17. Cont. Separation of viral gradient Phe / Chl treatment to get dsRNA