Thermal cycler dice real time system lite

Sepasol^®-RNA I Super G (Nacalai Tesque, Kyoto, Japan) was used to extract total RNA. RNA (1 µg) was used to convert cDNA by ReverTra Ace^® (TOYOBO, Osaka, Japan). cDNA was used as a template for quantitative-PCR using SYBR^® Premix Ex Taq^TM II (Tli RNaseH Plus) (Takara Bio, Kusatsu, Japan) and the following primers: 5′-GCCTGGGAACAACCGGAAGGTG-3′ and 5′-GGGTGCCAGTTCCACCCGTTC-3′ for the 148-bp fragment of ICAM-1 [41 (link)] and 5′-GGACATCCGCAAAGACCTGTA-3′ and 5′-GCTCAGGAGGAGCAATGATCT-3′ for the 143-bp fragment of β-actin [42 (link)]. PCR was performed with Thermal Cycler Dice^® Real Time System Lite (Takara Bio, Kusatsu, Japan) under the following conditions: 94 °C for 3 min, followed by 45 cycles of 95 °C for 5 s, 58 °C for 30 s, and 72 °C for 30 s. The quantity of initial mRNA was calculated from primer-specific standard curves.

Relative mRNA levels of the BTN2, HSP30, CUR1-FLAG, GIC2-FLAG, and YUR1-FLAG genes were determined by performing quantitative reverse transcription-PCR (qRT-PCR). Total RNA was extracted from yeast cells by using a method described by Schmitt et al. (1990) (link). RNA obtained was reverse transcribed to cDNA by using ReverTra Ace qPCR RT Master Mix FSQ-201 (Toyobo, Osaka, Japan), according to the manufacturer’s instructions. Quantitative PCR was performed using Thermal Cycler Dice Real Time System Lite (Takara Bio Inc., Shiga, Japan) and SYBR^® Premix Ex Taq^TM II (Takara Bio Inc., Shiga, Japan). Comparison of mRNA expression levels was performed by normalizing the mRNA level of each gene to that of ACT1 (Takahashi et al., 2011 (link)). The mRNA level was expressed as the ratio of normalized mRNA level of the target gene to that of the reference gene. Oligonucleotide sequences of primers used in qRT-PCR are listed in Table 2.

The gut bacteria in sand flies were quantified by qPCR with TB Green Fast Mix (TOYOBO Co., Ltd., Osaka, Japan) using a Thermal Cycler Dice Real Time System Lite (Takara Bio Inc., Shiga, Japan). The bacterial 16S rRNA gene was used as the target, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the reference. The primer sequences were 5ʹ-ACHCCTACGGGDGGCWGCAG-3ʹ (16S-q-337F) and 5ʹ-GTDTYACCGCGGYTGCTGGCAC-3ʹ (16S-q-514R) for the amplification of bacterial 16S rRNA gene, and 5ʹ-TTCGCAGAAGACAGTGATGG-3ʹ (Lugapdh-q-F) and 5ʹ-CCCTTCATCGGTCTGGACTA-3ʹ (Lugapdh-q-R), and 5ʹ-CGACTTCAACAGCA ACTCCCACTCTTCC-3ʹ (Phgapdh-q-F) and 5ʹ-TGGGTGGTCCAGGGTTTCTTACT CCTT-3ʹ (Phgapdh-q-R) for gapdh gene [35 , 36 (link)]. Relative quantities of 16S rRNA genes were determined by ∆∆Ct method using GAPDH as the reference.