3.2 Production
of Labeled dsRNA A488
Work described in this subchapter can be completed within 2 days.
This includes total RNA extraction, cDNA synthesis, and amplification followed by purification of the target template flanked by T7
promoter sequences on day 1, and production, washes, and quantification of the labeled dsRNA A488 on day 2.
1. For total RNA extraction, start by collecting 1-week-old mycelium (Vl41) grown in a PDB flask in a 50 mL Falcon tube and
centrifuge at 3000 Â g for 10 min.
2. Discard the supernatant and homogenize the pellet using mortar and pestle in liquid nitrogen.
3. Collect the homogenized sample into 2 mL Eppendorf SafeLock Tubes (50 to 100 mg of tissue). Samples can be immediately used or stored at À80
C for a longer period.
4. Under a chemical fume hood add 1 mL of TRIzol reagent to
the 2 mL collection tube, mix well, and incubate at RT for
5 min.
5. Add 200 μL of chloroform to the sample mix, shake vigorously
for 15 s, incubate at RT for 2–3 min followed by centrifugation
at 12,000 Â g for 15 min at 4
C. After centrifugation, the
TRIzol mix will separate into a lower red phenol-chloroform
phase, an interphase, and a colorless upper aqueous phase.
6. Place the upper aqueous phase carefully in a new 2 mL collection tube and, always under a chemical fume hood, add 0.5 mL
isopropanol to the mix. Incubate at RT for 10 min followed by
centrifugation at 12,000 Â g for 10 min at 4
C.
7. Discard the supernatant and wash the pellet with 1 mL of 75%
ethanol. Briefly vortex the sample and centrifuge it at 7500 Â g
for 5 min at 4
C. Discard the ethanol solution and allow the
pellet to air-dry for 5 min.
8. Finally, dissolve the pellet in 50 μL RNase-free water by heating
in a water bath at 55–60
C for 15 min and measure the total
RNA concentration via spectrophotometric analysis (absorption wavelength 260/230 nm). Proceed with the cDNA synthesis or store the RNA at À80
C (see Note 5).
9. For removal of genomic DNA mix 2 μg of the RNA with 1 μL
of DNase I (1 U/μL), 1 μL of DNase buffer (10Â), and 8 μL of
RNase-free water to reach the total volume of 10 μL. Incubate
at 37
C for 30 min.
10. Add 2 μL of EDTA (50 mM) and heat the sample at 65
C for
10 min in a water bath to stop any DNase activity.
11. For cDNA synthesis, set up a 20 μL total volume reaction by
placing ~1 μg of the RNA (5 μL from the previous step), 1 μL
reverse transcriptase, 5 μL reverse transcriptase buffer (1Â final
concentration), and RNase-free water. Incubate the reaction in
232
Matteo Galli et al.
of Labeled dsRNA A488
Work described in this subchapter can be completed within 2 days.
This includes total RNA extraction, cDNA synthesis, and amplification followed by purification of the target template flanked by T7
promoter sequences on day 1, and production, washes, and quantification of the labeled dsRNA A488 on day 2.
1. For total RNA extraction, start by collecting 1-week-old mycelium (Vl41) grown in a PDB flask in a 50 mL Falcon tube and
centrifuge at 3000 Â g for 10 min.
2. Discard the supernatant and homogenize the pellet using mortar and pestle in liquid nitrogen.
3. Collect the homogenized sample into 2 mL Eppendorf SafeLock Tubes (50 to 100 mg of tissue). Samples can be immediately used or stored at À80
C for a longer period.
4. Under a chemical fume hood add 1 mL of TRIzol reagent to
the 2 mL collection tube, mix well, and incubate at RT for
5 min.
5. Add 200 μL of chloroform to the sample mix, shake vigorously
for 15 s, incubate at RT for 2–3 min followed by centrifugation
at 12,000 Â g for 15 min at 4
C. After centrifugation, the
TRIzol mix will separate into a lower red phenol-chloroform
phase, an interphase, and a colorless upper aqueous phase.
6. Place the upper aqueous phase carefully in a new 2 mL collection tube and, always under a chemical fume hood, add 0.5 mL
isopropanol to the mix. Incubate at RT for 10 min followed by
centrifugation at 12,000 Â g for 10 min at 4
C.
7. Discard the supernatant and wash the pellet with 1 mL of 75%
ethanol. Briefly vortex the sample and centrifuge it at 7500 Â g
for 5 min at 4
C. Discard the ethanol solution and allow the
pellet to air-dry for 5 min.
8. Finally, dissolve the pellet in 50 μL RNase-free water by heating
in a water bath at 55–60
C for 15 min and measure the total
RNA concentration via spectrophotometric analysis (absorption wavelength 260/230 nm). Proceed with the cDNA synthesis or store the RNA at À80
C (see Note 5).
9. For removal of genomic DNA mix 2 μg of the RNA with 1 μL
of DNase I (1 U/μL), 1 μL of DNase buffer (10Â), and 8 μL of
RNase-free water to reach the total volume of 10 μL. Incubate
at 37
C for 30 min.
10. Add 2 μL of EDTA (50 mM) and heat the sample at 65
C for
10 min in a water bath to stop any DNase activity.
11. For cDNA synthesis, set up a 20 μL total volume reaction by
placing ~1 μg of the RNA (5 μL from the previous step), 1 μL
reverse transcriptase, 5 μL reverse transcriptase buffer (1Â final
concentration), and RNase-free water. Incubate the reaction in
232
Matteo Galli et al.