Quantitative real-time PCR analysis (qRT-PCR) was performed to verify the RNA-seq
gene expression profile. Nine unigenes related to anthraquinone biosynthesis
were selected for qRT-PCR using the same RNA samples used in RNA-seq. cDNAs were
synthesized with total RNA with reverse transcriptase M-MLV (Takara) using oligo
d(T)18 primers. The primer sequences of these genes are shown in
Table S1 .
qRT-PCR was performed on a LightCycler 480 II Real-Time PCR Cycler (Roche,
Switzerland) with SybrGreen qPCR Master Mix (BBI, China). The 18S rRNA of
R. tanguticum was selected as an internal reference. The 20
µl reaction mixture used contained 2 µl of cDNA, 10 µl of SYBR green qPCR master
mix, 0.8 µl of forward and reverse primers and 7.2 µl of ddH2O. All
reactions were carried out in a LightCycler 480 II Real-Time PCR Cycler (Roche,
Switzerland) with the following conditions: an initial step of 3 min at 95 ℃
for, followed by 5 s at 95 ℃ and 30 sat 60 ℃ for 45 cycles. Each reaction was
conducted with three biological and three technical repeats. The relative gene
expression level for each sample was calculated with the 2-∆∆Ctmethod (Livak and Schmittgen, 2001 (link)).
gene expression profile. Nine unigenes related to anthraquinone biosynthesis
were selected for qRT-PCR using the same RNA samples used in RNA-seq. cDNAs were
synthesized with total RNA with reverse transcriptase M-MLV (Takara) using oligo
d(T)18 primers. The primer sequences of these genes are shown in
qRT-PCR was performed on a LightCycler 480 II Real-Time PCR Cycler (Roche,
Switzerland) with SybrGreen qPCR Master Mix (BBI, China). The 18S rRNA of
R. tanguticum was selected as an internal reference. The 20
µl reaction mixture used contained 2 µl of cDNA, 10 µl of SYBR green qPCR master
mix, 0.8 µl of forward and reverse primers and 7.2 µl of ddH2O. All
reactions were carried out in a LightCycler 480 II Real-Time PCR Cycler (Roche,
Switzerland) with the following conditions: an initial step of 3 min at 95 ℃
for, followed by 5 s at 95 ℃ and 30 sat 60 ℃ for 45 cycles. Each reaction was
conducted with three biological and three technical repeats. The relative gene
expression level for each sample was calculated with the 2-∆∆Ctmethod (Livak and Schmittgen, 2001 (link)).