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7 protocols using trnzol a

1

Extracting Total RNA from Insect Antennae

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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.
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2

Gastric Cancer Tissue and Plasma RNA Extraction

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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 −80C. Blood samples (5 mL) were collected from all subjects in EDTA tubes and centrifuged at 3,000 rpm for 10 min at 4C, then the plasma was cautiously collected and also keep it at −80C 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.
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3

Quantitative Analysis of Protein-Gene Expression

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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)].
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4

Quantification of Sodium-Dependent Phosphate Transporters

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Total RNA was extracted using TRNzol-A + (TIANGEN, Beijing, China). The concentration of total RNA was estimated by a spectrophotometer (Ultrospec 2100 pro, GE Healthcare, Chicago, IL, USA), and the purity was determined by agarose gel electrophoresis. Five hundred nanagrams of total RNA were reverse transcribed into cDNA using the Fast Quant RT Kit (with gDNase) (TIANGEN). qPCR was conducted using the iCycler iQ5 system. The specific primers for NaP-IIb, type III sodium-dependent phosphate cotransporter-1,2 (PiT-1, 2) and β-actin are listed in Table 2. β-actin was used as internal reference gene. Relative gene expression was calculated using the 2-ΔΔCt method [24 (link)]. All the samples were analysed in triplicate, and the operational program for qPCR strictly followed the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) [25 (link)].
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5

Plasmid and Genomic DNA Isolation, Southern Blot, and RNA Extraction

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Plasmid DNA was isolated with the SanPrep plasmid mini kit (Sangon, B518191) and S. scitamineum genomic DNA was extracted using the method described previously (Yan et al., 2016b (link)). For Southern blot analysis, DNA samples (3–5 μg) digested with appropriate restriction enzymes were separated by electrophoresis and blotted to Hybond N+ membrane. Standard hybridization and detection protocols were performed using the method of DIG DNA labeling and detection kit (Roche, 11093657910). Total RNA was isolated with TRNzol-A+ (Tiangen, DP421) and first-strand cDNA was synthesized using the Revert AidFirst Strand cDNA Synthesis Kit (Thermo Fisher Scientific, K1621).
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6

Gene Expression Analysis of M. separata

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Antennae of 1-day- and 5-day-old 25 adult M. separata (males and females separately) were collected after 3.5 h in the dark. Samples from each group were immediately homogenized in TRNzol-A+ (TIANGEN Biotech, Beijing, China) on ice, and total RNA was extracted according to the manual. The concentration and purity of total RNA were determined by using a spectrophotometer NanoDrop2000 (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 were used in the following experiments. Extracts were treated with DNase I (Takara, Kusatsu, Shiga, Japan) to remove any DNA. RNA extractions were replicated three times.
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7

Quantitative gene expression analysis in chicken

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Total RNA was extracted using TRNzol-A+ (TIANGEN, Beijing, China). The concentration of total RNA was estimated by spectrophotometer (Ultrospec 2100 pro, GE Healthcare), and the purity was determined by agarose gel electrophoresis. 500 ng of total RNA was reversely transcribed into cDNA using the Fast Quant RT Kit (with gDNase) (TIANGEN). qPCR was conducted using the iCycler iQ5 system. The specific primers for NaP-IIb, PiT-1, PiT-2, and β-actin were listed in Table 2. β-actin, was used as internal reference gene. Relative gene expression was calculated using the 2 -ΔΔCt method [23] . All the samples were analyzed in triplicate and operational program for qPCR strictly followed the MIQE [24] .
Table 2 Primer sequences of chicken NaP-IIb, PiT-1, 2, and β-actin Gene Primer sequence (5'-3') Accession number
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