Computational protocol: Beneficial Effects of Ethyl Pyruvate through Inhibiting High-Mobility Group Box 1 Expression and TLR4/NF-κB Pathway after Traumatic Brain Injury in the Rat

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Protocol publication

[…] The levels of HMGB1 and TLR4 mRNA expression were determined by RT-PCR. Total RNA was extracted with TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer's instructions. Total cDNA was synthesized from the isolated total RNA using a reverse transcriptional system. Briefly, 5 μg of total RNA was reverse transcribed using 0.5 μg oligo(dT)15 U avian myeloblastosis virus reverse transcriptase (Biouniquer Technology CO, LTD). The cDNA was then amplified by PCR. The forward and reverse primers were as follows: 5′-ATGGGCAAAGGAGATCCTA-3′ and 5′-ATTCATCATCATCATCTTCT-3′ for HMGB1 (646 bp), 5′-TTGCCTTCATTACAGGGACTT-3′ and 5′-CAGAGCGGCTACTCAGAAACT-3′ for TLR4 (179 bp), and 5′-GCCATGTACGTAGCCATCCA-3′ and 5′-GAACCGCTCATTGCCGATAG-3′ for β-actin (375 bp) [, ]. PCR amplification was performed for 35 cycles of 30 seconds at 94°C, 50 seconds at 55°C, and 50 seconds at 72°C, followed by a final step of 7 minutes at 72°C. PCR products were detected by agarose gel electrophoresis in 2% NuSieve agarose gels (FMC, USA) and visualized by ethidium bromide staining. The intensity of the bands was quantified using ImageJ software, and the ratios of each gene product normalized to β-actin product were considered as the expression of each gene. [...] Densitometric quantification and analysis of the PCR, EMSA, and Western blot bands were done with ImageJ software. SPSS 16.0 was used for the statistical analysis (SPSS, Inc., Chicago, IL, USA). All data are expressed as mean ± SEM. The data were subjected to one-way ANOVA followed by Tukey's post-hoc test. Statistical significance was accepted with P < .05. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Rattus norvegicus
Diseases Brain Edema, Brain Injuries, Cerebrovascular Trauma