Relative expression software tool 2009

Tables 1 and 2 show the patients and healthy control subjects adjusted for potential confounding factors such as demographic variables and tumor characteristics. Based on relative expression of miR-21 as a target gene versus miR-16 as a reference gene, the up-regulated expression of miR-21 was measured using the ΔΔCT method[29 (link)] and Relative Expression Software Tool 2009 (Qiagen, Hilden, Germany). The Analyses of variance (ANOVA), Kruskal-Wallis, and Mann-Whitney statistic tests were used to evaluate the differences of miRNA expression levels in serum or stool samples. As a diagnostic test, Receiver Operating Characteristic (ROC) curve analysis was applied to determine the sensitivity and the specificity of miR-21 expression in serum and stool of CRC patients. In addition, internal validation was performed using the BCa bootstrap method to accurately estimate the ROC curves and optimal cut-off values. The method was used to discriminate CRC patients from healthy control subjects, as well as patients with TNM stages III-IV from stages I-II. Data analysis was performed by using MedCalc®version 13.1.2.0 (Acacialaan 22, 8400 Ostend, Belgium).

The groups were compared by one-way analysis of variance (ANOVA) followed by a specific post hoc test. Statistical analysis was performed using Relative Expression Software Tool 2009 (Relative Expression Software Tool, Qiagen, Hilden, Germany), which is based on the Pair Wise Fixed Reallocation Randomization Test© (Pfaffl et al., 2002). Differences were considered statistically significant for *p < 0.05, **p < 0.01, and ***p < 0.001.

Gene expression of the most common loci and genes known to be involved in the VISA mechanism, including graTSR, vraTSR, walKR, agr RNAIII, sceD, pbpB, and, fmtA were quantified in wild-type VAN-S and mutant CHG_Van-I strains using the Reverse Transcription quantitative PCR (RT-qPCR) method. The gyrA gene was used as an internal control gene.
For this purpose, RNA was extracted (GeneAll Hybrid-R™ RNA isolation kit, Korea) from mid-logarithmic phase cultures, then treated with DNase I (Thermo Fisher Scientific, USA) and reversed to cDNA (Yekta Tajhiz Azma cDNA synthesis kit, Iran). The RT-qPCR was carried out in a Rotor-Gene Q (Qiagen, Germany) with the cDNA template in the final concentration of 5 ng, SYBR Green (Ampliqon, Denmark), and previously designed primers (Pishgam Biotech, Iran) [3 (link), 11 (link)–14 (link)].
The RT-qPCR experiment was independently performed three times. Relative expression data were analyzed using the Relative Expression Software Tool (REST) 2009 (v2.0.13; Qiagen, USA) by the ∆∆Ct method. The pairwise fixed randomization test with 2000 iterations (as randomization number) in a rest standard mode was used.

To validate the microarray results, the expression levels of 10 selected genes in the cultured cells treated with GO (compared with cultured cells treated with buffer) were quantified by real-time reverse transcription-polymerase chain reaction (RT-qPCR). cDNA was synthesized from 400 ng of total RNA using the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Amplification primers were designed using the Clone Manager Suite (Sci-Ed Software, Morrisville, USA). RT-qPCR assays were carried out using the LightCycler^®480 and LightCycler^®480 FastStart SYBR Green I Master (Roche Diagnostics GmbH, Germany). All assays were run in triplicate. Quantification cycles (Cq) were calculated using the second derivative method (LightCycler^®480 Software, Roche). The fold change in gene expression levels, corrected by efficiency, was analyzed using Relative Expression Software Tool (REST 2009) (Qiagen; [57 (link)]). The expression data were normalized to the polymerase (RNA) II (DNA directed) polypeptide A (POLR2A) and ribosomal protein L29 (RPL29) genes, which in a RefFinder algorithm-based selection were the most stable among the 4 candidate reference genes tested. All experiments were performed according to the MIQE guidelines [58 (link)].