Transcriptional Profiling of Lipid-Related Genes

Total RNA extracted from the liver and colon tissues was reverse-transcribed to cDNA using a SuperScript™ first-strand synthesis system for reverse transcription PCR (RT-PCR) (Invitrogen, United States). RT-PCR was performed on a LightCycler96 (Hangzhou Bioer Technology, Share-Holding Co.) using iQ SYBR Green Supermix (BIO-RAD, United States). The relative expression of lipid synthesis-related genes (Scd1, PPARγ [34 (link)]), lipid hydrolysis-related genes (Adrb3, Lipe, Pnpla2 [35 (link)]), lipid oxidation-related genes (Cpt2, Acox1, Ppargc1a [35 (link)], PPARα [36 (link)]), lipid transport gene (Fabp5 [37 (link)]), intestinal permeability-related genes (Occludin, ZO-1, Muc5 [38 (link)]), and inflammation-related genes (CD14 [39 (link)], TLR2, TLR4, NLRC4, and MCP-1 [40 (link)]) were adjusted with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene. Relative quantification was calculated using the 2^−∆∆Ct method. The primer sequences for RT-PCR can be found in Supplementary Table 1.

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Wang R.R., Zhang L.F., Chen L.P., Wang J.Y., Zhang L., Xu Y.S., Yang P.L., Ji G, & Liu B.C. (2021). Structural and Functional Modulation of Gut Microbiota by Jiangzhi Granules during the Amelioration of Nonalcoholic Fatty Liver Disease. Oxidative Medicine and Cellular Longevity, 2021, 2234695.

Publication 2021

SuperScript™ first-strand synthesis system for reverse transcription PCR iQ SYBR Green Supermix

Cdna Colon Expression genes Genes Glyceraldehyde 3 phosphate dehydrogenase Housekeeping gene Hydrolysis Inflammation Intestinal Lipid Lipid synthesis Mcp 1 Occludin Permeability Ppargc1a Pparγ Primer Reverse transcription Sybr green Synthesis Tissues Tlr2

Corresponding Organization : Longhua Hospital Shanghai University of Traditional Chinese Medicine

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Variable analysis

independent variables

Tissue type (liver and colon)

dependent variables

Relative expression of lipid synthesis-related genes (Scd1, PPARγ)
Relative expression of lipid hydrolysis-related genes (Adrb3, Lipe, Pnpla2)
Relative expression of lipid oxidation-related genes (Cpt2, Acox1, Ppargc1a, PPARα)
Relative expression of lipid transport gene (Fabp5)
Relative expression of intestinal permeability-related genes (Occludin, ZO-1, Muc5)
Relative expression of inflammation-related genes (CD14, TLR2, TLR4, NLRC4, MCP-1)

control variables

Housekeeping gene (GAPDH) used for normalization

controls

No positive or negative controls were explicitly mentioned.

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