Sybr premix ex taq 2 tli rnaseh plus master mix

Cellular RNA was isolated with Trizol reagent, and first strand cDNA was synthesized with the Prime Script RT kit (Takara Inc.) from 500 ng total RNA of each sample. Two μl of the resulting cDNA (5-fold dilution) was subjected to real-time PCR using SYBR Premix Ex Taq II (Tli RNase H Plus) master mix (Takara Inc). Forward and reverse PCR primers are listed in Supplementary Table 1 and were in different exons to avoid amplification of genomic DNA. Melting curve analysis was done to confirm single PCR products. We used the 2^−ΔCt method^{79 (link)} to calculate mRNA expression of each gene relative to β-actin after initial confirmation that neither loss of PINK1 nor treatment with LPS/IFN-γ altered the expression of the internal standard β-actin (p > 0.05, t-test).

Antennae, proboscises, maxillary palps, thoraxes, legs, abdomens, heads (without antennae, proboscises, and maxillary palps), and wings (50 pairs of each sex) were collected for total RNA extraction using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The first strand cDNA template was synthetized with One-Step gDNA removal and cDNA Synthesis kit (TransGen, Beijing, China) including oligo dt-primer according to product manual recommendations. The primers of CpunPBP2, CpunPBP5 and reference gene (β-actin, accession number JX119014) for real-quantitative PCR (qPCR) were designed using Primer premier 5.0 program (Premier Biosoft International, Palo Alto, CA, USA; Table S1). qPCR were conducted on ABI 7500 fast real-time PCR system (Applied Biosysterm, USA). Each amplification reaction was performed with 20 μL volume using SYBR Premix Ex Taq II (Tli RNaseH Plus) master mix (Takara-Bio, Shiga, Japan) under the following conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. To check reproducibility, each test sample was done in triplicate technical replicates and three biological replicates. Relative quantification was analyzed using the comparative 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)). The relative expression levels in different tissues were calculated with the transcript level of the female antennae used as the calibrator.

The expression levels of ORs were examined in male and female antennae using qRT-PCR. Total RNA was extracted as described above. First-strand cDNAs were synthetize followed One-Step gDNA removal and cDNA Synthesis kit (TransGen Biotech, China) with oligo dT-primer following the kit manual. The primers for qPCR were designed using Primer premier5.0 program (Table S1). qRT-PCR were performed on ABI 7500 fast real-time PCR system (Applied Biosysterm, USA) in a reaction volume of 20 μL using SYBR Premix Ex Taq II (Tli RNaseH Plus) master mix (Takara-Bio, Shiga, Japan), according to the manufacturers’ instructions. Two-step program were performed as follows, 95 °C for 30 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. For each gene, three biological replications were performed with each biological replication measured in three technique replications. Relative quantification was analyzed using the comparative 2^−ΔΔCT method, with the housekeeping genes β-actin (accession number JX119014) as the reference gene.