3. Add 10 ml of sterile 10% glycerol to each agar culture (see
Note 9). Rub the surface of a sporulated culture with a sterile
spatula. Harvest the spores from the dishes with a sterile pipette
and pass through a borosilicate (size-1 pore) filter on top of a
sterile 250-ml flask (see Note 10). Transfer the filtered spores
to a 50-ml Falcon tube.
4. Centrifuge the spore solution at 2000 Â g for 20 min at 4
C.
Remove the supernatant and resuspend the pellet of spores in
1 ml of sterile water.
5. Count the spores with a Neubauer chamber.
6. Optimize concentration and time of addition of doxycycline
(Dox) for gene induction (see Note 11).
7. Inoculate 100 ml of DG minimal medium with 10
6 spores, two
flasks for each strain, one to induce the Tet-on system and
another one as control. Incubate for 3 days in darkness at
30
C in a shaker at 150 rpm. After 48 h add 10 μg/ml Dox
(see Note 11) to induce the Tet-on system. As controls, strains
were cultured without Dox.
8. Filter the mycelia through a sterile filter paper, wash them with
sterile water and freeze in liquid nitrogen. Store the samples at
À80
C until use.
3.3.2 RNA Extraction
1. Take approximately 100 μl of 0.5-mm zirconia/silica beads and
introduce in a 2-ml microfuge tube with cap. As a reference, fill
until reaching the cone of the tube (see Note 12).
2. Add 450 μl of lysis (RLT) buffer from RNeasy Plant Mini Kit,
previously supplemented with β-mercaptoethanol, and add
2–3 mg of frozen mycelium to each 2-ml tube.
3. Grind the mycelium in a FAST-PREP24 in two disruptions
pulses of 30 s at 4 m/s.
4. Extract RNA using the RNeasy Plant Mini Kit according to the
manufacturer’s protocol and quantify RNA (e.g., in a NanoDrop ND-1000 spectrophotometer). Check RNA integrity by
agarose gel electrophoresis.
3.3.3 Synthesis of cDNA
1. Add 2.5 μg of RNA in a volume of 7 μl and 1 μl of 10Â buffer
(included in the kit) to a PCR tube. Add 10 U of DNase I and
incubate for 15 min at 25
C using the thermocycler.
2. Stop the reaction by adding 1 μl of 50 mM EDTA (pH 8.0) and
heating at 65
C for 10 min.
3. Add 1 μl of RNA-free double distilled H 2 O and quantify the
RNA (e.g., using a NanoDrop ND-1000 spectrophotometer).
4. Use Transcriptor first-strand cDNA synthesis kit for retrotranscription. To the previous 10-μl RNA samples (steps 1–3) add
1 μl of anchored-oligo (dT) and 2 μl RNA-free distilled H 2 O
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
353
Note 9). Rub the surface of a sporulated culture with a sterile
spatula. Harvest the spores from the dishes with a sterile pipette
and pass through a borosilicate (size-1 pore) filter on top of a
sterile 250-ml flask (see Note 10). Transfer the filtered spores
to a 50-ml Falcon tube.
4. Centrifuge the spore solution at 2000 Â g for 20 min at 4
C.
Remove the supernatant and resuspend the pellet of spores in
1 ml of sterile water.
5. Count the spores with a Neubauer chamber.
6. Optimize concentration and time of addition of doxycycline
(Dox) for gene induction (see Note 11).
7. Inoculate 100 ml of DG minimal medium with 10
6 spores, two
flasks for each strain, one to induce the Tet-on system and
another one as control. Incubate for 3 days in darkness at
30
C in a shaker at 150 rpm. After 48 h add 10 μg/ml Dox
(see Note 11) to induce the Tet-on system. As controls, strains
were cultured without Dox.
8. Filter the mycelia through a sterile filter paper, wash them with
sterile water and freeze in liquid nitrogen. Store the samples at
À80
C until use.
3.3.2 RNA Extraction
1. Take approximately 100 μl of 0.5-mm zirconia/silica beads and
introduce in a 2-ml microfuge tube with cap. As a reference, fill
until reaching the cone of the tube (see Note 12).
2. Add 450 μl of lysis (RLT) buffer from RNeasy Plant Mini Kit,
previously supplemented with β-mercaptoethanol, and add
2–3 mg of frozen mycelium to each 2-ml tube.
3. Grind the mycelium in a FAST-PREP24 in two disruptions
pulses of 30 s at 4 m/s.
4. Extract RNA using the RNeasy Plant Mini Kit according to the
manufacturer’s protocol and quantify RNA (e.g., in a NanoDrop ND-1000 spectrophotometer). Check RNA integrity by
agarose gel electrophoresis.
3.3.3 Synthesis of cDNA
1. Add 2.5 μg of RNA in a volume of 7 μl and 1 μl of 10Â buffer
(included in the kit) to a PCR tube. Add 10 U of DNase I and
incubate for 15 min at 25
C using the thermocycler.
2. Stop the reaction by adding 1 μl of 50 mM EDTA (pH 8.0) and
heating at 65
C for 10 min.
3. Add 1 μl of RNA-free double distilled H 2 O and quantify the
RNA (e.g., using a NanoDrop ND-1000 spectrophotometer).
4. Use Transcriptor first-strand cDNA synthesis kit for retrotranscription. To the previous 10-μl RNA samples (steps 1–3) add
1 μl of anchored-oligo (dT) and 2 μl RNA-free distilled H 2 O
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
353