2.2 Total RNA
Extraction, DNase
Treatment, and cDNA
Synthesis
1. Fungal mycelium (Vl41).
2. 50 mL Falcon tubes.
3. Cooled laboratory centrifuge.
4. Mortar and pestle.
5. Water bath.
6. Spectrophotometer.
7. 1.5 and 2 mL Eppendorf Safe-Lock Tubes.
8. Liquid nitrogen.
9. Filter sheet (pore size: 22–25 μm).
10. RNase- and DNase-free water.
11. TRIzol (guanidinium thiocyanate, store at RT) (see Note 2).
12. Chloroform (use it under chemical hood).
13. Isopropanol.
14. 70% Ethanol.
15. DNase I-free RNase (1 U/μL).
16. 10Â DNase I buffer: 100 mM Tris–HCl, 25 mM MgCl 2 ,
1 mM CaCl 2 , pH 7.5.
17. 50 mM Ethylenediaminetetraacetic acid (EDTA), pH 7.
18. Reverse transcriptase.
19. 10Â Reverse transcriptase buffer: 100 mM Tris–HCl, 500 mM
KCl, 1% Triton-X-100, pH 9.0.
20. Thermocycler.
21. PCR mix: 1.5 mM MgCl 2 , Taq polymerase (0.5 U) with
specific buffer, 4 mM deoxynucleotides (dNTPs), and
0.1–0.5 μM of primers.
22. Agarose powder.
23. Tris/borate/EDTA buffer: 109.03 g/l (0.9 M) Tris base,
55.0 g/l (0.9 M) boric acid, 9.31 g/l (0.025 M) EDTA.
24. DNA ladder for gel electrophoresis.
25. Horizontal gel electrophoresis apparatus and power supply.
26. Ethidium bromide.
27. PCR cleanup kit.
28. Membrane-binding solution: 5.5 M Guanidine hydrochloride,
20 mM Tris–HCl, pH 6.6.
29. Membrane wash solution: 20 mM NaCl, 2 mM Tris–HCl, 80%
ethanol V/V, pH 7.5.
30. Silica-membrane spin column (for DNA purification).
Labeling of dsRNA for Fungal Uptake Detection Analysis
229