Trnzol a

Antennae of 4-d-old adults were used. A total of 25 adults (males and females separately) were collected after 3.5 h of the dark cycle. Antennae samples from each group were immediately homogenized in TRNzol-A+ (TIANGEN Biotech, Beijing, China) on ice, and total RNA was extracted according to the manufacturer’s instructions. The concentration and purity of the total RNA were determined by using a NanoDrop2000 spectrophotometer (ThermoFisher, Waltham, MA, USA). RNA with an A260/A280 ratio between 1.75–2.05, an A260/A230 ratio > 1, and a concentration > 400 ng/μL was used for the experiments. Total RNA was treated with DNase I (Takara, Kusatsu, Shiga, Japan) to remove any genomic DNA. RNA extractions were performed in triplicate.

All the GC specimens including tissues and blood, were obtained from patients who received surgical resection for GC at Shanghai General Hospital affiliated of Shanghai Jiaotong University. Before RNA extraction, all specimens were snap-frozen instantly and stored at −80^◦C. Blood samples (5 mL) were collected from all subjects in EDTA tubes and centrifuged at 3,000 rpm for 10 min at 4^◦C, then the plasma was cautiously collected and also keep it at −80^◦C until use. Total RNA extraction from tissues and plasma samples used TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and TRNzol A+ (TIANGEN, Beijing, China). miRNA used miRcute Serum/plasma miRNA isolation kit (TIANGEN, Beijing, China) according to the manufacturer’s protocols. After adding denaturing solution (Ambion) for normalization of the sample-to-sample variation, 1 ul of synthetic external control (1 umol/L; TIANGENN) was spiked into each sample.

To further understand the relationship between proteins and their encoding genes, qPCR was run for proteins of differential hepatic abundance at the mRNA level. Specific primers for target genes of the identified proteins were designed using the primer BLAST of NCBI and nucleotide information in GenBank (Additional file 2: Table S2). Total RNA was prepared from the liver of control and treated groups using TRNzol-A+ (TIANGEN, Beijing, China). RNA quality and concentration were detected using spectrophotometer (Ultrospec 2100 pro, GE Healthcare) and agarose gel electrophoresis. cDNA synthesis with 5 μg of RNA was performed using the Fast Quant RT Kit (with gDNase) (TIANGEN). qPCR was conducted using the iCycler iQ5 system. The PCR was performed in a 20-μL reaction system containing 1 μL of cDNA, 0.5 μL of each primer (10 μM), 10 μL of Super Real PreMix (SYBR Green) (TIANGEN) and 8.2 μL of water. The fold-change was calculated using the IQTM5 software (Bio-Rad) with the 2 ^−ΔΔCt method [25 (link)]. All operation for qPCR was followed by the MIQE [26 (link)].