Thunderbird next sybr qpcr mix kit

An RNA extraction solution, Isogen II (Nippon Gene Co., Tokyo, Japan), was used to isolate total RNA from tissue pieces of the lung stored in RNA Save solution. The cDNA was synthesized by incubating 2–4 µg of total RNA with 100 U of ReverTra Ace reverse transcriptase (Toyobo Co., Osaka, Japan) with a mixture of 20 pmol random hexamers pdN6 and 5 pmol oligo-dT(15) primers (Takara Bio Inc., Kusatsu, Japan). A qPCR instrument, StepOnePlus (Applied Biosystems/Life Technologies Co., Carlsbad, CA, USA), was employed for cDNA measurement with Thunderbird Next Sybr qPCR Mix Kit (Toyobo Co.). PCR fragments for each cDNA were prepared separately and purified by gel electrophoresis before quantitative analysis. The DNA sequences were confirmed by Fasmac Co., Ltd. (Atsugi, Japan). The extracted DNA fragments were used as standards for quantification. PCR conditions were 2 min of initial denaturation followed by 40 cycles of 5 s at 95 °C and 35 s at 60 °C. The measured mRNA levels were normalized with reference to the β-actin mRNA levels. Specific primer sets for genes are listed in Table 1.

The expression levels of Ada2b, RpL32, Drosomycin, CG6675, and CG33462 were determined using RT-PCR. To prepare fly samples, Ml or Sa suspension (OD = 1) was injected into flies as described above. The flies were collected at 4, 8, and 16 h post-challenge infection. Total RNA was extracted from three flies for each sample, and three samples were analyzed as biological replicates for each experimental condition. Complementary DNA (cDNA) was synthesized from total RNAs using ReverTra Ace (Toyobo), following the manufacturer’s instructions. RT-PCR analysis was performed using the Thunderbird Next SYBR qPCR Mix kit (Toyobo) or FastStart DNA Master SYBR Green I (Roche) with Light Cycler 96 (Roche). The primers used for RT-PCR analysis are listed in S6 Table. The relative gene expression was quantified using the R program. The expression data were statistically compared using ANOVA, followed by Tukey’s HSD post-hoc test with the R program.

Internodal segments (8 samples/set; Fig. 1) were placed in a 2.0-mL tube with a zirconia bead (diameter, 5 mm), frozen in liquid nitrogen, and crushed in a TissueLyser II (Qiagen). Total RNA was extracted with an RNeasy Plant Mini Kit (Qiagen) following the supplied instructions. cDNA was synthesized from 100 ng of total RNA using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). Transcript levels were determined by qPCR using a Thunderbird NEXT SYBR qPCR Mix kit (Toyobo). qRT-PCR was performed with a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). The expression of ELONGATION FACTOR1 (EF1) was used as a reference. The primer sets used are listed in Table S1. Statistical analysis of relative expression was carried out in IBM SPSS Statistics 26.0 software (IBM SPSS Inc., Armonk, NY, USA). Following assessment of the equality of variances by ANOVA, multiple comparison was conducted by Tukey’s honestly significant difference (Tukey’s HSD). P values less than 0.05 were considered statistically significant.