Computational protocol: Intestinal anti-inflammatory activity of Ground Cherry (Physalis angulata L.) standardized CO2 phytopharmaceutical preparation

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Protocol publication

[…] Male Wistar rats (180-200 g) obtained from ANILAB - Animais de Laboratório, Paulínia, São Paulo (Brazil), were housed in standard environmental conditions (21 °C, 60%-70% humidity) with 12-h light/dark cycle and air filtration. Animals had free access to water and food (Biobase). Experimental protocols met the ‘‘Guidelines of Animal Experimentation’’ approved by the Ethical Committee for Animal Research (Protocol number 042/04-CEEA), Institute of Biosciences, Universidade Estadual Paulista (UNESP). [...] Colon samples (100 mg) were collected and stored in -80 °C until use for analyses of genes: GAPDH, β-actin, HPRT, HSP70, heparanase, NF-κB, mitogen-activated protein kinase (MAP)K1, MAPK3, MAPK6, MAPK9, MUC1, MUC2, MUC3, and MUC4. For the homogenization, we used 1 mL of Trizol® (Invitrogen-Life Technologies, Carlsbad, CA, United States) and a Polytron homogenizer. The total RNA extraction was made according to the Trizol® manufacturer’s protocol. The purity was determined by A260/A280 ratio using a Nanodrop 2000 (Thermo Scientific, Waltham, MA, United States). After that, 1 μg of the total RNA of colon tissue samples was incubated with DNAse I (1 U/mg RNA; Invitrogen), and then reverse transcribed with SuperScript® III (200 U/mL; Life Technologies) and oligo-d (T) primer. Primers for targets and reference genes were designed based on the rat sequences and using the IDT primer quest software (http://www.idtdna.com/primerquest/Home/Index; Invitrogen). Relative real-time RT-PCR analysis was performed with a StepOne Plus™ (Applied Biosystems Inc., Foster City, CA, United States) using Power SYBR Green PCR Master Mix® (Life Technologies) for all the genes. Amplification efficiencies for target and reference genes were similar. The primer sequences, fragment size, annealing temperature, primer concentration, NCBI reference sequence and sample concentration for each gene are shown in Table .Reactions were optimized to provide maximum amplification efficiency for each gene. PCR was performed in 25 μL reaction volumes in duplicate, in a MicroAmp® Fast Optical 96-Well Reaction Plate, 0.1 mL (Applied Biosystems Inc.), and the specificity of each PCR product was determined by melting curve analysis. Negative controls (water replacing cDNA) were run in every plate.The relative expression of each target gene was calculated using the ∆∆Ct method with efficiency correction[]; the control was a cDNA sample from each cell type analyzed. To select the most stable reference gene for detailed analyses, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin and HPRT amplification profiles were compared using the RefFinder software (http://www.leonxie.com/referencegene.php?type=reference). All gene expression analysis was performed with β-actin as the reference gene for colon tissue. […]

Pipeline specifications

Software tools Biobase, PrimerQuest
Applications Miscellaneous, qPCR
Organisms Rattus norvegicus
Diseases Amino Acid Metabolism, Inborn Errors, Inflammatory Bowel Diseases
Chemicals Carbon Dioxide, Glutathione