Total RNA was extracted and purified by standard Trizol method and using the Allprep Kit (Qiagen). All RNA quality and quantity were determined using RNA6000 labchip/Agilent 2100 bioanalyzer.
Label
FAM
Label protocol
As per manufacturer (ABI).
Hybridization protocol
n/a
Scan protocol
n/a
Description
test
Data processing
RQ Manager integrated in software from ABI was used to normalize the entire signal generated. Expression level (as fold change) was calculated when both tumor and normal samples had signals in the assays using DataAssist software v2.0 (Life Technologies, http://www.lifetechnologies.com/about-life-technologies.html). Signals for miRNA that showed either in tumor only or normal only were dropped from analysis. Fold change was calculated using the 2 -ΔΔCT method. In the present study, the data are presented as fold change in the target gene expression in tumors normalized to the internal control gene (MammU6) and relative to the normal tissue control (matched normal as calibrator). Results of the real-time PCR data are represented as CT values, with CT defined as the threshold cycle number of PCRs at which amplified product was first detected. The average CT was calculated for both the target gene and MammU6 and the ΔCT was determined as (the mean of up to three CT values for the target gene) minus (the mean of the CT values for U6). The ΔΔCT represented the difference between the paired tissue samples, as calculated by the formula ΔΔCT = (ΔCT of tumor - ΔCT of normal). The N-fold differential expression in the target gene of a tumor sample compared to its normal sample counterpart was expressed as 2 -ΔΔCT. For each case, the frequency of dysregulated miRNAs was calculated as the number of dysregulated miRNAs divided by the total number of miRNAs that showed signals in both tumor and normal. The criteria used to call an miRNA dysregulated were fold changes ≥ 2 or ≤ 0.5.
