Computational protocol: Enhanced Gastrointestinal Expression of Cytosolic Malic Enzyme (ME1) Induces Intestinal and Liver Lipogenic Gene Expression and Intestinal Cell Proliferation in Mice

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Protocol publication

[…] RNA was extracted from individual jejunum, ileum, distal colon and liver samples (n = 7−9 mice/group/tissue) with TRIzol (Life Technologies, Grand Island, NY). One microgram of RNA was reverse-transcribed to obtain cDNA using the iScript cDNA synthesis kit (Bio-Rad; Hercules, CA). Expression of target genes was assayed by qRT-PCR using Bio-Rad iTaq SYBR Green Supermix. PCR primers () were obtained from Integrated DNA Technologies, Inc. (Coralville, IA). Target mRNA abundance was normalized to a factor derived from the geometric mean of expression values for mouse β-Actin (Actb), Cyclophilin A (Ppia) and TATA box binding protein (Tbp), calculated using GeNorm . Student’s t-test was used to compare variables between groups (SigmaPlot 12; Systat Software, Inc.; Chicago, IL). To detect expression of the ME1 transgene in the colon and small intestine by conventional RT-PCR, a primer pair was used to amplify a segment spanning the SV40 poly A and ME1 coding regions [forward primer (Me1): 5′-AAT GAT TCG GTC TTC CTC ACC-3′, and reverse primer (SV40): 5′-CAG ACA TGA TAA GAT ACA TTG ATG AGT T-3′] of the transgene construct. […]

Pipeline specifications

Software tools geNorm, SigmaPlot
Applications Miscellaneous, qPCR
Organisms Mus musculus, Rattus norvegicus, Homo sapiens
Diseases Diabetes Mellitus
Chemicals Bromodeoxyuridine, Cholesterol, Glucose, NADP