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By comparing the ion features in the assayed samples to the reference libraries, namely, the Human Metabolomics Database (HMDB) and KEGG, 106 metabolites were finally identified.
Relative expression was calculated by the comparative cycle threshold method and U6 microRNA was used as reference gene to normalise all RNA samples, because it is constant in the assayed samples by miR-profiling and quantitative RT-PCR analysis as previously reported.
All protein samples were dissolved in the assayed buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT.
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Compounds were diluted ten-fold in 1% (v/v) DMSO and used in the assays.
The solutions were stored at −20 °C before they were used in the assays.
Negative controls were included in the assays wherever required.
The concentrations reported are final concentrations in the assays.
The second-instar larvae were used in the assays.
The peptide concentration was 2 μM in the assays.
Internal controls were included in the assay.
Two different mobile phases were employed in the assay procedure.
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