Fastrna pro red kit

About 20 mg of T. reesei mycelium was harvested. Total RNA was extracted using the FastRNA Pro Red Kit (MPbio, U. S. A.), according to the manufacturer’s instructions. Reverse transcription was performed with 1000 ng of total RNA using TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen, China), according to the manufacturer’s instructions. For qPCR, the TransStart TipTop Green qPCR SuperMix (TransGen, China) was used with 200 nM of forward and reverse primers (see Supplemental Material Table S-4) and 1 μl of 10-fold diluted cDNA in a final volume of 20 μl. For hpt transcription analysis, a SYBR green assay with reference to a small GTPase gene (sar1) was performed. Thermocycling was performed in an ABI StepOne Plus thermocycler (Applied Biosystems, USA).

The levels of gene-specific mRNA were assessed using RT-qPCR as per our previous study [54 (link)]. In brief, total RNA was carefully extracted from frozen mycelia using a FastRNA Pro Red Kit (MPbio, Irvine, CA, USA) as per the manufacturer’s instructions. Total RNA (500 ng) was reverse-transcribed and cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA eraser (TaKaRa, Japan), according to the manufacturer’s instructions. qPCR was performed using an ABI StepOne thermocycler (Applied Biosystems, Foster City, CA, USA) and the TransStart TipTop Green qPCR SuperMix (TransGen, Shanghai, China) with 200 nM of forward and reverse primers (Additional file 1: Table S1). Thermal cycling was conducted under the following conditions: an initial denaturation step at 95 °C, followed by 40 amplification cycles of 5 s at 94 °C and 60 s at 64 °C. For transcription analysis, an SYBR green assay with reference to the sar1 gene was performed [57 (link)]. Melt curves were obtained after each RT-qPCR run to confirm the specificity of amplification and the absence of primer dimers.

The levels of gene-specific mRNA were assessed using RT-qPCR, according to our previous study, with some modification [59 (link)]. In brief, the total RNA of 50 mg fresh weight cells was extracted using a FastRNA Pro Red Kit (MPbio, Irvine, CA, USA), according to the manufacturer’s instructions. Synthesis of cDNA from total RNA was performed using the PrimeScript RT Reagent Kit with gDNA eraser (TaKaRa, Japan) as per the manufacturer’s instructions. For RT-qPCR, the TransStart TipTop Green qPCR SuperMix (TransGen, Shanghai, China) was used with 200 nM of forward and reverse primers (see Additional file 1: Table S1). Gene transcription was analyzed using SYBR green assays. Transcription levels of target genes were normalized to that of the sar1 gene [60 (link)]. Thermocycling was performed in an ABI StepOne Plus thermocycler (Applied Biosystems, Foster City, CA, USA).

Total RNA was isolated as previously described by Noguchi et al. [40 (link)]. The conidia (1.0 × 10⁶ cells/mL) were inoculated and precultured in Vogel’s liquid medium containing 1.2% glucose for 22 h at 25 °C on a reciprocating shaker (135 rpm) and then, the growing hyphae were transferred to fresh Vogel’s medium containing glucose or maltose and cultured for 2 h at 25 °C. The resultant mycelia were harvested by filtration using an aspirator, and frozen in liquid nitrogen until RNA isolation. Total RNA was isolated from each sample using a FastRNA Pro Red kit (MP Biomedicals, Tokyo, Japan). Each RNA sample (1 µg of total RNA) was subjected to cDNA synthesis and quantitative real-time reverse transcription PCR (RT-qPCR) using the LightCycler system (Roche Diagnostics, Tokyo, Japan), as described by Yamashita et al. [41 (link)]. The mRNA expression of the target genes was quantified using Universal ProbeLibrary (Roche Diagnostics, Tokyo, Japan). Primers and probes used in this study are summarized in Table S3. The relative gene expression value was calculated by comparing the threshold cycle (Cp), with actin used as the reference gene in the RT-qPCR analysis. A minimum of three biological replicates were performed for each experiment.

RNA was extracted using the FastRNA Pro Red Kit (MP Biomedicals, Santa Ana, CA, USA). cDNA was synthesized with TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen). The levels of gene-specific mRNA were assessed by RT-qPCR on an ABI StepOne Plus thermocycler (Applied Biosystems, Foster City, CA, USA). The primers are described in Additional file 1: Table S1. The cycling conditions comprised 30 s initial denaturation and polymerase activation at 95 °C, followed by 40 cycles of 5 s at 95 °C and 60 s at 64 °C. Threshold cycle (Ct) values and PCR efficiency rates were used to calculate relative expression quantities by the ABI software. Transcript levels of target genes were normalized to sar1 expression (Steiger et al. 2010 (link)) by the 2^−ΔΔCt method.

After 35 days of cultivation, colonies were collected as described in Section 2.2, and RNA was extracted from the biomass using the FastRNA Pro RED Kit (MP Biomedicals, Santa Ana, CA, USA). For each condition and strain, three replicates of total RNA were extracted following the manufacture’s protocol. The quality and quantity was measured using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and a Qubit 2.0 (Life Technologies, Carlsbad, CA, USA).

The total RNA of each sample was extracted by using the FastRNA Pro Red kit (MP Biomedicals, Santa Ana, CA, USA) and purified by using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNA products obtained from the samples were quantified and qualified by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo FisherScientific Inc.), and 1% agarose gel electrophoresis before next-generation sequencing of library preparations. RNA extraction, quantification, and qualification were each conducted more than three time, followed by mixing for further library preparation. Approximately 1 μg of extracted total RNA was used for library preparation, based on the manufacturer’s protocol of the NEBNext Ultra RNA Library Prep kit for Illumina. The library sequencing was conducted on the Illumina HiSeq X10 sequencing platform.