Computational protocol: Heat Shock Protein 27 is down regulated in Ballooned Hepatocytes of Patients with Nonalcoholic Steatohepatitis (NASH)

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Protocol publication

[…] Total RNA was prepared from a sub-sample of 30 liver biopsy specimens in which nucleic acids were available to perform molecular studies; 14 patients with NAFLD without ballooning degeneration and 16 patients with NASH and ballooned hepatocytes were included in the molecular analysis.RNA was purified from liver tissue using phenol extraction step method, with an additional DNAse digestion. For RT-PCR, 1 to 3 μg of total RNA was reverse-transcribed using random hexamers and Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Promega, Wisconsin, USA). Real-time PCR was performed for quantitative assessment of mRNA expression in a StepOne Plus Real-Time PCR Systems (Applied Biosystems, CA 94404, USA). All the real-time PCR reactions were run in triplicate.The mRNA abundance of target genes was normalized to the amount of a housekeeping gene (RPL19) to carry out comparisons between the groups. The selection of the housekeeping gene was based on the exploration of the most stable reference gene for testing liver mRNA expression among other housekeeping genes tested before starting this experiment. The geNorm program was used to identify the appropriate reference control in our samples.The mRNA levels were expressed as the ratio of the estimated amount of the target gene relative to the RPL19 mRNA levels using fluorescence threshold cycle values (Ct) calculated for each sample, and the estimated efficiency of the PCR for each product was expressed as the average of all sample efficiency values obtained. The specificity of amplification and absence of primer dimers were confirmed using the melting curve analysis at the end of each run. The primer sequences are shown in . [...] Quantitative data were expressed as mean ± standard deviation (SD) unless otherwise indicated. Since a significant difference in the SD was observed between the groups in most of the variables and the distribution was significantly skewed in most cases, we chose to be conservative and assessed the differences between the groups using nonparametric Mann–Whitney U or Kruskal–Wallis tests. For the same reasons, correlation between two variables was done with the use of Spearman’s rank correlation test. A multivariate logistic regression analysis considering covariates as independent variables and histological features (ballooning degeneration, lobular inflammation and fibrosis, absent = 0, present = 1) as the dependent variable was performed separately on the whole group of patients. Significance was accepted at the P < 0.05 level. The CSS/Statistica program package version 6.0 (StatSoft, Tulsa, OK, USA) was used in these analyses unless indicated otherwise.For plasma glucose levels, we constructed the receiver operating characteristic (ROC) curve and calculated the area under the ROC curve to evaluate the predictive power for liver histology using the free Web-based tool ROCCET (http://www.roccet.ca/ROCCET/) and the corresponding analysis for difference between AUROC was done by comproc subroutine as implemented in STATA 10.1 (Statacorp, College Station, TX, USA). […]

Pipeline specifications

Software tools geNorm, Statistica
Applications Miscellaneous, qPCR
Organisms Homo sapiens
Diseases Nerve Degeneration, Shock, Non-alcoholic Fatty Liver Disease
Chemicals Cholesterol, Glucose, Triglycerides