Nmr suite 4

The spectra were phased, baseline-corrected, and referenced to TSP (at 0.0 ppm) as well as quantified and qualified using a commercial software package (Chenomx NMR suite 4.6, Chenomx, Inc., Edmonton, Alberta, Canada) with the Chenomx 500-MHz (pH 6–8) library. The spectra data were pre-processed using normalization and scaling to remove possible bias arising due to sample handling and sample variability. Normalization (by sum) was performed in order to minimize possible differences in concentration between samples. In addition, the spectral region from 5.0 to 4.7 ppm containing the residual water resonance was excluded, and all subsequent analysis used centred scaling.
After data pre-processing, a univariate statistical analysis (Student′s t-test) was used to verify the significance of difference in metabolite levels between the CHH dsRNA-injected group and the saline-injected group. p<0.05 was considered to indicate a statistically significant difference. These significantly changed metabolites were further analyzed by heuristic methods of dimension reduction (principal component analysis [PCA]) using commercially available software (SIMCA-P 11.5; Umetrics, Umeå, Sweden), and all subsequent analysis used centred scaling. The number of PCA components was calculated using an auto-fit model in SIMCA-P (Umetrics).

Experimental treatment protocol, efficacy and specificity of gene silencing by CHH dsRNA, as well as NMR setting parameters, data acquisition and processing, and statistical analysis (Student's t-test) have been described in a previous study [34 (link)]. Briefly, double-stranded RNAs were produced using an in vitro transcription reaction driven by T7 promoter and T7 RNA Polymerase. Animals were treated with either saline injection (SAI) or CHH dsRNA injection (CHH DSI), and, at designated time points (24, 48, 72 hours after treatment), tissues were dissected under stereo-microscopes (SMZ745, Nikon) using fine scissors and used for a quantitative real-time PCR (the eyestalk ganglia) or a dual phase extraction procedure (the muscle and hepatopancreas). CHH dsRNA, but not green fluorescence protein dsRNA, effectively and significantly decreased CHH gene expression in the eyestalk ganglia at the time points examined. Aqueous phase extracts of the muscle and hepatopancreas were subjected to ¹H Nuclear magnetic resonance (NMR) analysis (Varian Inova 500 MHz NMR spectrometer). The spectra were analyzed using a commercial software package (Chenomx NMR suite 4.6, Chenomx, Inc., Edmonton, Alberta, Canada) with the Chenomx 500-MHz (pH 6±8) library.