(supplied in the kit) and incubate at 65
C for 10 min. The
result is the Template-primer mix.
5. Prepare the RT mix and add it to the Template-primer mix
following the supplier’s instructions of the Kit.
6. Calculate theoretic cDNA concentration using the equation:
Theoretic [cDNA] ¼ [RNA]  Vi  E/Vf. Being [RNA]:
RNA concentration after DNAse treatment. Vi: initial volume,
E: retrotranscriptase efficiency, and Vf: final volume. In our case
[cDNA] theoretic ¼ [RNA] Â 10 Â 0.7/20.
7. To calculate what volume of water must be added to the sample
to get a final concentration of 25 ng/μl use the equation:
V ¼ [cDNA] Â Vf/ (cc–Vf) ¼ [cDNA] Â 20/(25–20).
3.3.4 Analysis by
Quantitative RT-PCR
1. In a 1.5-ml microcentrifuge tube, prepare the qPCR mix the
LightCycler 480 SYBR Green I Master with the selected primers for carS gene. Prepare a primer mix adding 10 μl of
100 μM primer RtFfcarS-1F primer and 10 μl of 100 μM
primer RtFfcarS-1F in 80 μl of double distilled H 2 O. For the
RT mix (see Note 13), add 2.6 μl of vial 2 (PCR-grade water),
5 μl of vial 1 (Master mix 2Â) and 0.4 μl of 10 μM primer mix.
Fig. 3 Expression of carS gene controlled by the Tet-on system. Levels of mRNA
of the gene carS under the control of the Tet-on promoter controlled by
doxycycline in strain SG262 and compared to F. fujikuroi IMI 58289 wild type
(WT), and carS mutant (SG39). Strains were cultivated in 100 ml of DG medium
in 500-ml flasks during 48 h at 30
C and 150 rpm in darkness. Tet-on
expression was induced with 10 μg/ml of doxycycline (Dox) and incubated for
24 h; non-induced cultures were grown in parallel. RNA was extracted (see
Subheading 3.3.2), converted to cDNA (see Subheading 3.3.3) and analyzed by
qPCR (see Subheading 3.3.4). β-tubulin gene was used as a reference for
relative expression
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Julia Marente et al.
C for 10 min. The
result is the Template-primer mix.
5. Prepare the RT mix and add it to the Template-primer mix
following the supplier’s instructions of the Kit.
6. Calculate theoretic cDNA concentration using the equation:
Theoretic [cDNA] ¼ [RNA]  Vi  E/Vf. Being [RNA]:
RNA concentration after DNAse treatment. Vi: initial volume,
E: retrotranscriptase efficiency, and Vf: final volume. In our case
[cDNA] theoretic ¼ [RNA] Â 10 Â 0.7/20.
7. To calculate what volume of water must be added to the sample
to get a final concentration of 25 ng/μl use the equation:
V ¼ [cDNA] Â Vf/ (cc–Vf) ¼ [cDNA] Â 20/(25–20).
3.3.4 Analysis by
Quantitative RT-PCR
1. In a 1.5-ml microcentrifuge tube, prepare the qPCR mix the
LightCycler 480 SYBR Green I Master with the selected primers for carS gene. Prepare a primer mix adding 10 μl of
100 μM primer RtFfcarS-1F primer and 10 μl of 100 μM
primer RtFfcarS-1F in 80 μl of double distilled H 2 O. For the
RT mix (see Note 13), add 2.6 μl of vial 2 (PCR-grade water),
5 μl of vial 1 (Master mix 2Â) and 0.4 μl of 10 μM primer mix.
Fig. 3 Expression of carS gene controlled by the Tet-on system. Levels of mRNA
of the gene carS under the control of the Tet-on promoter controlled by
doxycycline in strain SG262 and compared to F. fujikuroi IMI 58289 wild type
(WT), and carS mutant (SG39). Strains were cultivated in 100 ml of DG medium
in 500-ml flasks during 48 h at 30
C and 150 rpm in darkness. Tet-on
expression was induced with 10 μg/ml of doxycycline (Dox) and incubated for
24 h; non-induced cultures were grown in parallel. RNA was extracted (see
Subheading 3.3.2), converted to cDNA (see Subheading 3.3.3) and analyzed by
qPCR (see Subheading 3.3.4). β-tubulin gene was used as a reference for
relative expression
354
Julia Marente et al.