Faststart dna master sybr green 1

Total RNA was extracted from the DG using the ReliaPrep RNA Miniprep System (Promega), and subjected to reverse transcription with Superscript VILO (Invitrogen), and followed by real-time PCR on a LightCycler (Roche) using the Fast Start DNA Master SYBR Green I (Roche). Expression levels of target genes were normalized to the levels of 18S rRNA. Primer sequences are shown in Table 1. The specificity of each primer set was confirmed by checking the product size by gel electrophoresis.

Total cellular RNA was isolated from PBMCs, BFCs or CD14+-isolated monocytes using the High Pure RNA Isolation Kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions and 1 μg was reverse transcribed with random hexanucleotides and AMV reverse transcriptase (Roche) in a final volume of 20 μL for 1 h at 42 °C. For skin biopsies, RNA was extracted using TriReagent^® (Molecular Research Center, Inc. Cincinnati, OH, USA) according to the manufacturer’s instructions, and 500 ng was used for reverse transcription.
The expression of human IL15 and IL15RA genes was analyzed by PCR amplification of 1 μL aliquots of the resulting cDNAs in a total volume of 15 μL, in a Light Cycler (Roche) with FastStart DNA Master SYBR Green I (Roche). Standard curves for target mRNA expression were generated by amplifying 10-fold serial dilutions of known quantities of the specific PCR product. Quantification of target gene expression was obtained using Light Cycler system software version 4.05 (Roche, Mannheim, Germany). B2M was used as a housekeeping reference gene, except for experiments involving skin biopsies, in which ARF5 was used as a reference. Relative units estimated from the quantification (Rq) represent the ratio between specific mRNA molecules/μL cDNA and molecules of B2M (or ARF5) mRNA/μL cDNA.
The primers used are shown in Table S3 in the Supplementary Materials.

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.

Total RNA was extracted from the cells using the RNeasy Plus Mini Kit (QIAGEN K.K., Tokyo, Japan). Reverse transcription was performed on 0.5~1.0 µg of total RNA using the PrimeScript^® RT reagent Kit (TAKARA Bio. Inc. Shiga, Japan). Real-time quantitative PCR was performed on cDNA aliquots with the FastStart DNA Master SYBR Green I and the LightCycler (Roche Diagnostics, Mannheim, Germany). The sequences of the oligonucleotide primer pairs used to amplify the mRNA in the mouse and human cells are shown in Table 1. PCR amplification began with a 10 min pre-incubation step at 95°C, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 62°C for 10 s, and elongation at 72°C (Table 1). The relative concentration of PCR product derived from the target gene was calculated using the LightCycler System software. A standard curve for each run was constructed by plotting the crossover point against the log concentration. The concentration of target molecules in each sample was then calculated automatically by reference to this curve (r = −1.00), and the results were standardized to the expression of β-actin. The specificity of each PCR product was assessed by melting curve analysis.