protocol provided by the manufacturer, mix in a nuclease-free
microtube placed on ice 4 μL T7 reaction buffer (5Â), 4 μL
labeling mix (5Â), 0.25 μL RNase inhibitor (40 U/μL), and
0.25 μL T7 RNA polymerase (200 U/μL), and add linearized
T7-template-DNA (up to 1 μg) and RNase-free water to a total
volume of 20 μl. Incubate the mix at 37
C for 30 min in a
water bath in darkness.
21. Remove the DNA template by DNase (see procedure steps 9–
11) and purify the probe through silica-based membrane columns: transfer the labeled dsRNA A488 mix to a silica-based
membrane column, incubate at RT for 1 min, and centrifuge
at 13,000 Â g for 30 s. Wash two times with 700 μL of RNA
wash buffer followed each time by centrifugation at 13,000 Â g
for 30 s. Ensure the complete removal of the RNA wash buffer
via centrifugation at 13,000 Â g for 2 min. Finally transfer the
column into a RNase-free 1.5 mL tube, elute the labeled
dsRNA A488 with 25 μL of DNase/RNase-free water, and centrifuge at 13,000 Â g for 30 s. Perform these steps in a semidark room to avoid degradation of the probe due to light (see
Note 9).
22. Quantify the labeled dsRNA concentration via spectrophotometric analysis (e.g., using NanoDrop nanospectrometry
equipment) and conserve the mix at À80
C.
3.3 Labeled dsRNA
Feeding to Fungus
and Detection
of Fluorescent Signal
1. Isolate conidia from 5- to 7-day-old fungal broth culture in
sterile conditions (see Notes 10 and 11). To separate the
mycelia from the conidia, filtrate the broth culture through
two layers of a sterilized filter sheet (pore size: 22–25 μm)
positioned on a sterilized glass funnel. Collect the conidia in a
50 mL Falcon tube and count the conidia with a counting
chamber under a bright-field microscope. Set the final concentration to 20,000 conidia per ml of PDB. Conidia can be stored
in PDB at 4
C up to 2 days after isolation (see Note 12) or at
À80
C in deionized water for a longer period. In the case of a
low conidia yield, smash mycelia bulk bodies prior to filtration.
This technique will free the conidia trapped in the mycelia. Use
freshly isolated vital conidia for the in vitro assay.
2. Into each well of a flat-bottom microplate (96-well), add
500 freshly isolated conidia, 250–500 ng of labeled dsRNA,
and PDB up to 100 μL of total volume. For the control sample,
place an equal volume of nuclease-free water as for labeled
dsRNA added into the test samples.
3. Place the microplate on a shaker at low speed (20 g) for 2 days
at RT (see Note 13). During this time, keep the microplate in a
darkroom, to avoid the loss of fluorescence of labeled dsRNA
due to light.
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Matteo Galli et al.
microtube placed on ice 4 μL T7 reaction buffer (5Â), 4 μL
labeling mix (5Â), 0.25 μL RNase inhibitor (40 U/μL), and
0.25 μL T7 RNA polymerase (200 U/μL), and add linearized
T7-template-DNA (up to 1 μg) and RNase-free water to a total
volume of 20 μl. Incubate the mix at 37
C for 30 min in a
water bath in darkness.
21. Remove the DNA template by DNase (see procedure steps 9–
11) and purify the probe through silica-based membrane columns: transfer the labeled dsRNA A488 mix to a silica-based
membrane column, incubate at RT for 1 min, and centrifuge
at 13,000 Â g for 30 s. Wash two times with 700 μL of RNA
wash buffer followed each time by centrifugation at 13,000 Â g
for 30 s. Ensure the complete removal of the RNA wash buffer
via centrifugation at 13,000 Â g for 2 min. Finally transfer the
column into a RNase-free 1.5 mL tube, elute the labeled
dsRNA A488 with 25 μL of DNase/RNase-free water, and centrifuge at 13,000 Â g for 30 s. Perform these steps in a semidark room to avoid degradation of the probe due to light (see
Note 9).
22. Quantify the labeled dsRNA concentration via spectrophotometric analysis (e.g., using NanoDrop nanospectrometry
equipment) and conserve the mix at À80
C.
3.3 Labeled dsRNA
Feeding to Fungus
and Detection
of Fluorescent Signal
1. Isolate conidia from 5- to 7-day-old fungal broth culture in
sterile conditions (see Notes 10 and 11). To separate the
mycelia from the conidia, filtrate the broth culture through
two layers of a sterilized filter sheet (pore size: 22–25 μm)
positioned on a sterilized glass funnel. Collect the conidia in a
50 mL Falcon tube and count the conidia with a counting
chamber under a bright-field microscope. Set the final concentration to 20,000 conidia per ml of PDB. Conidia can be stored
in PDB at 4
C up to 2 days after isolation (see Note 12) or at
À80
C in deionized water for a longer period. In the case of a
low conidia yield, smash mycelia bulk bodies prior to filtration.
This technique will free the conidia trapped in the mycelia. Use
freshly isolated vital conidia for the in vitro assay.
2. Into each well of a flat-bottom microplate (96-well), add
500 freshly isolated conidia, 250–500 ng of labeled dsRNA,
and PDB up to 100 μL of total volume. For the control sample,
place an equal volume of nuclease-free water as for labeled
dsRNA added into the test samples.
3. Place the microplate on a shaker at low speed (20 g) for 2 days
at RT (see Note 13). During this time, keep the microplate in a
darkroom, to avoid the loss of fluorescence of labeled dsRNA
due to light.
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Matteo Galli et al.