Computational protocol: Chronic unpredictive mild stress leads to altered hepatic metabolic profile and gene expression

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Protocol publication

[…] Liver tissues were collected and stored in liquid nitrogen until use. Each sample of total RNA was extracted from 100 mg tissues using the TRIzol Reagent. RNA integrity was analyzed on a 1.2% agarose gel. RNA quantity was determined using a NanoDrop 2000C Spectrophotometer (Thermo Scientific). 1 μg of RNA was reverse-transcribed with a PrimeScript™ RT reagent Kit (TaKaRa) for cDNA synthesis and genomic DNA removal. The qPCRs were performed according to the instructions of the SYBR premix Ex Taq™ II kit (TliRNaseH Plus) (TaKaRa) and carried out in triplicate using the CFX96™ real-time PCR detection system (Bio-Rad). Reaction conditions were: 30 s at 95 °C, followed by 40 cycles of 95 °C for 5 s, and 60 °C, 30 s. Gene-specific primers were designed using online primer designing tools primer-blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesis in Invitrogen. The primer sequences are listed in supporting information (). The lengths of amplifications are between 100 bp and 250 bp. GAPDH was chosen as an endogenous control in this study. Standard deviations were calculated from three PCR replicates. The specificity of amplification was assessed by dissociation curve analysis, and the relative abundance of genes was determined using the comparative Ct method as suggested by the CFX-manager software (Bio-Rad). […]

Pipeline specifications

Software tools Primer-BLAST, CFX Manager
Application qPCR
Organisms Rattus norvegicus
Diseases Chemical and Drug Induced Liver Injury