Faststart universal sybr green master rox supermix

For reverse transcription-quantitative PCR (RT-qPCR) analysis, strains were grown in a 2 l fermenter with K & R medium, and the mycelium was harvested at 24, 48, and 72 h. Total RNA of M. circinelloides was extracted with Trizol after grinding under liquid N₂ and reverse-transcribed using the Prime ScriptRT reagent kit (Takara) according to the manufacturer’s instructions. RT-qPCR was performed using primers GLqPCR-F/R (Additional file 1: Table S1) on LightCycler 96 Instrument (Roche Diagnostics GmbH, Switzerland) with FastStart Universal SYBR Green Master (ROX) Supermix (Roche) according to the manufacturer’s instruction. The mRNA expression level was normalized to levels of 18S rRNA, and the results were expressed as relative expression levels. The data were quantified by the method of 2^−ΔΔCt.

For RNA extraction, Mc-55 was grown in a 2 L fermenter with 1.5 L K&R media, and biomass were collected at 3, 24, 48, and 72 h. Total RNA was extracted by using TRIzol after disruption of biomass in pestle and mortar, using liquid nitrogen as described previously (22 (link)). RNA was reverse transcribed using the Prime ScriptRT reagent kit (Takara Biotechnology, Dalian Co., Ltd, Dalian, China) according to the instructions of the manufacturer. To investigate the expression levels of the canthaxanthin biosynthesis gene bkt, real-time quantitative PCR (RT-qPCR) was conducted using specifically designed primers (Supplementary Table S2) on Light Cycler 96 Instrument (Roche Diagnostics GmbH, Switzerland) with FastStart Universal SYBR Green Master (ROX) Supermix (Roche) according to the instructions of the manufacturer. The mRNA expression level was normalized to levels of actin gene and the results were determined as relative expression levels. The data were quantified by the method of 2–ΔΔ^Ct.

Total RNA was isolated from rice leaves using TRIzol Reagent (TransGen) according to the manufacturer's instructions. Approximately 3 μg of RNA sample was subjected to RNase-free DNaseI (Invitrogen) treatment and reverse transcribed using M-MLV Reverse Transcriptase (Invitrogen) with Oligo(dT)15. Quantitative RT–PCR was performed in a ViiA 7 Real-Time PCR system (Applied Biosystems) using FastStart Universal SYBR Green Master (Rox) superMIX (Roche). Ubiquitin was used as a reference gene in the qRT–PCR experiments. The measurements were obtained using the relative quantification method. The significant difference was analyzed statistically by student's t test or one-way analysis of variance (ANOVA). Primer pairs for qRT-PCR analysis are listed in Table S3.

Strains were grown in a 2 L fermenter with modified K&R medium, and the mycelium was harvested at 3, 6, 12, 24, 48, and 72 h. Extraction of total RNA of M. circinelloides was done by using TRIzol after disruption of biomass in pestle and mortar, using liquid nitrogen. In order to investigate mRNA levels of four key genes of the carotenogenic pathway, RT-qPCR was conducted. Reverse transcription of RNA was performed by using the Prime ScriptRT reagent kit (Takara Biotechnology, Dalian Co., Ltd, Dalian, China) according to the manufacturer’s instructions. Primers were designed according to RT-qPCR requirement (Supplementary Table S1), and RT-qPCR was performed by using these primers on Light Cycler 96 Instrument (Roche Diagnostics GmbH, Switzerland) with FastStart Universal SYBR Green Master (ROX) Supermix (Roche) as instructed by the manufacturer. The mRNA expression level was normalized to the level of actin gene, and the results were expressed as relative expression levels. The data were quantified by the method of 2^−ΔΔCt [7 (link)].

According to the manufacturer’s instructions, the total RNA was extracted from rice different tissues using TRIzol reagent (Invitrogen). About 3 μg of RNA sample was processed by RNase-free DNaseI (Invitrogen) and reverse transcribed using M-MLV reverse transcriptase (Invitrogen) with Oligo(dT)15. Quantitative RT-PCR was carried out using Fast Start Universal SYBR Green Master (Rox) superMIX (Roche, Mannheim, Germany) in a ViiA 7 Real-Time PCR system (Applied Biosystems), according to the manufacturer’s introductions. Measurements were obtained using the relative quantification method. Actin was used as a reference gene in the qRT-PCR experiments. The experiment was designed with three biological replicates and three technical replicates per material. Error bars indicate standard error. The measurements were obtained using the relative quantification method. The significant difference was analyzed statistically by One-way ANOVA and Student’s t-tests. All primers for qRT-PCR analysis are listed in Supplementary Table S2.

Total RNA was extracted from leaves using an RNA extraction kit (TRIzol reagent, Invitrogen) following the manufacturer’s instructions. First-strand cDNA was reverse transcribed from DNase I-treated RNA with oligo(dT) as the primer. The expression of OsHAP5A, OsHAP5B, OsHAP3D and OsHAP3E was measured by quantitative real-time reverse transcription PCR (qRT-PCR). Approximately 3mg total RNA was reverse transcribed using M-MLV reverse transcriptase (Invitrogen) in a volume of 180 µl to obtain cDNA. The Ubiquitin gene was used as an internal control for the qRT-PCR. The primers used for qRT-PCR are listed in Supplementary Table S3. qRT-PCR was run in a total volume of 15 µl containing 3.6 µl of the reverse-transcribed product obtained as described above, 0.25mM gene-specific primers and 7.8 µl FastStart Universal SYBR Green Master (Rox) superMIX (Roche, Mannheim, Germany) on an Applied Biosystems ViiA 7 RT-PCR system, according to the manufacturer’s instructions. The relative quantification method was used to measure the quantity of qRT-PCR product. The qRT-PCR was performed using the following program: 95 °C for10min, then 40 cycles of 95 °C for 5s and 60 °C for 34s.