Rest2009 program

Three gels per condition were pooled, solubilized in Trizol (Invitrogen) and RNA isolated using the RNeasy Micro RNA kit with on column DNase I digestion as recommended by the manufacturer (Qiagen, Valencia, CA). RNA concentrations were determined using a BioRad Experion system with standard sensitivity RNA chips and 50 ng of total RNA was used to prepare cDNA using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). PCR reactions were carried out using iQ Supermix (BioRad) with primers specific for rat α-smooth muscle actin (α-SMA) (forward: 5′ GGAGTGATGGTT-GGAATGG 3′; reverse: 5′ ATGATGCCGTGTTCTATCG 3′) and control gene acidic ribosomal phosphoprotein (ARBP) (forward: 5′ TAGAGGGT-GTCCGCAATG 3′; reverse: 5′ GAAGGTGTAGTCAGTC-TCC 3′). Data were analyzed using the fold change method incorporating primer efficiency with the REST2009 program (Qiagen Inc., Valencia, CA).

Expression levels of genes were analyzed by ViiA7 System Software (Life Technologies) and statistical analysis was performed using SPSS v16.0 Software (SPSS Inc., Chicago, IL). Fold changes were calculated usig raw cycle threshold (Ct) data by the REST2009 program (Qiagen), which is routinely used for the determination of differences between different types of sample and control groups and considers both normalization to numerous reference genes and reaction efficiencies [23 (link)]. Then ratios of Ct values of genes of interest and mean value of reference genes were calculated and used for further statistical analyses. Differences in gene expression or methylation levels between tumor and control tissues were assessed by the nonparametric Mann-Whitney U-test. To evaluate associations of transcript levels with clinical data and other variables (Table 1), nonparametric tests (the Kruskal-Wallis, the Mann-Whitney, and the Spearman’s tests) were used.
DFI was evaluated by the Kaplan-Meier method and the Log Rank test was used for evaluation of the compared subgroups and combined groups of patients. Stage-adjusted analysis was performed by the Cox regression. All P-values were calculated from two-sided tests. P-values lower than 0.05 were considered statistically significant. The correction for multiple testing was applied according to Bonferroni.

The REST 2009 program (Qiagen, Germany) was used to quantify changes in the expression levels of genes and to determine statistical differences between the CCHF and control groups, and between CCHF fatal and survivor groups. Differences in gene expression were also analyzed between intermediate or high SGS risk score participants (Group 1) and low SGS risk score (Group 2). P values less than or equal to 0.05 were accepted as statistically significant. Descriptive analysis of patient characteristics was performed using the Statistical Package for the Social Sciences (SPSS) version 15 for Windows (SPSS Inc., Chicago, IL, USA).