Thermal cycler dice real time system tp850

Quantification of the bacterial 16S rRNA gene and fungal 18S rRNA gene was accomplished using real-time PCR. Bacterial DNA was quantified using the primers 1055f and 1392r and the TaqMan probe 16Staq1115 (Harms et al., 2003 (link)). Fungal DNA quantification was conducted using the primers FungiQuant-F and FungiQuant-R and the TaqMan probe FungiQuant-Prb (Liu et al., 2012 (link)). For fungi, universal fungal primers, and TaqMan probes covered the 1,199–1,549 S. cerevisiae numbering region of the 18S rRNA-encoding gene. Each reaction mixture was prepared in a total volume of 25 μL with 12.5 μL Premix Ex Taq (Probe qPCR, Takara Bio), 0.2 μM of each primer, 0.25 μM TaqMan probe, and 2 μL of standard or extracted DNA. For the assay, PCR amplification was performed in a Thermal Cycler Dice Real Time System (TP-850, Takara Bio, Otsu, Japan) under the conditions of initial denaturation for 30 s at 95°C followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. DNA standards for bacteria and fungi were prepared from serial dilutions of the pGEM-T Easy Vector (Promega) containing the 16S rRNA gene from Escherichia coli and the 18S rRNA gene from Cladosporium sp., respectively. Duplicate aliquots of the standards and the samples were included in each PCR run and all assays included a negative control without template DNA.

cDNA was synthesized from 5 μg of total RNA from each sample using the GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster City, CA, USA). The cDNA were amplified with KAPA SYBR FAST Universal qPCR kit (Kapa Biosystems, Wilmington, MA, USA) using the following primers (Additional file 1: Table S1). The quantitative RT-PCR (RT-qPCR) experiments were performed using a Thermal Cycler Dice Real Time System TP850 (Takara Bio, Otsu, Japan).

Quantitative polymerase chain reaction (qPCR) was performed with the cDNA derived in the previous section, THUNDERBIRD® SYBR Green (Toyobo, Osaka, Japan) and the primer sets in Table 2 on Thermal Cycler Dice Real Time System TP850 (TaKaRa, Shiga, Japan). The thermal cycling protocol of qPCR was as follows: initial denaturation at 95 ∘ C for 1 min and followed by 45 cycles of amplification. Each cycle consisted of 95 ∘ C for 10 s and 60 ∘ C for 30 s. The qPCR programme ended with dissociation curve analysis in the following order: 95 ∘ C for 15 s, 60 ∘ C for 30 s and 95 ∘ C for 15 s. The absolute quantity of each gene was derived from the second Derivative Maximum Method. Both the transcription levels of GbDHCR24-1 and GbDHCR24-2 were normalized to the that of β -actin. All datapoints were performed twice to confirm the reproducibility.