Computational protocol: Differential Influence of Inositol Hexaphosphate on the Expression of Genes Encoding TGF-β Isoforms and Their Receptors in Intestinal Epithelial Cells Stimulated with Proinflammatory Agents

Similar protocols

Protocol publication

[…] Detection of the expression of genes encoding TGF-β isoforms and their receptors was carried out using a qRT-PCR technique with a SYBR Green chemistry (SYBR Green Quantitect RT-PCR Kit, Qiagen) and Opticon DNA Engine Continuous Fluorescence detector (MJ Research) as described previously []. Oligonucleotide primers specific for TGF-β1, TGF-β2, TGF-β3, TβRI, TβRII, and TβRIII mRNAs were designed using Primer Express 2.0 software (PE Applied Biosystems, USA) (). The thermal profile for one-step RT-PCR was as follows: 50°C for 30 min for reverse transcription and 95°C for 15 min followed by 45 cycles at 94°C for 15 s, 55°C for 30 s, and 72°C for 45 s for amplification. Following RT-PCR, the samples were subjected to temperature ramp from 60°C to 95°C at the rate of 0.2°C/s with continuous fluorescence monitoring for melting curve analysis. Each gene analysis was performed in triplicate. A commercially available standard of β-actin (TaqMan DNA Template Reagent Kit, Applied Biosystems) was used to estimate the mRNA copy numbers of examined genes. The obtained results of mRNA copy number were recalculated per μg of total RNA. The expression level of examined genes in cultured cells was expressed as the fold change relative to the control. The value of fold change >1 reflects increased expression of the target gene, and a value of fold change <1 points to a decrease in the gene expression. Finally, specificity of RT-PCR reaction was confirmed by determining the characteristic temperature of melting for each amplimer and by 6% polyacrylamide gel (PAA) electrophoresis of RT-PCR products with their visualization using silver staining. [...] The results were collected based on three independent experiments. Statistical analysis was performed with the use of Statistica PL 9.0 software. All the results are expressed as means ± SD. Comparison of two data sets was performed by unpaired t-test. Significance level was assumed for P < 0.05. […]

Pipeline specifications

Software tools Primer Express, Statistica
Applications Miscellaneous, qPCR
Organisms Escherichia coli, Salmonella enterica subsp. enterica serovar Typhimurium
Chemicals Phytic Acid