miRNA quantification in plasma
Cell-free RNA molecules were extracted from the plasma using TRIzol reagent (Applichem, Düren, Germany). Briefly, 200 μl of the plasma sample was mixed with 800 μl TRIzol reagent, and homogenized. The mixture was added to 200 μl chloroform and incubated on ice for 5 min. Following centrifugation at 12,550 × g for 5 min, the upper RNA phase was transferred into 550 μl propanol containing tubes. Following centrifugation at 12,550 × g, the RNA containing pellet was washed with 75% ethanol, air-dried and dissolved in polymerase chain reaction (PCR)-grade water.
cDNA was synthesized using the miScript Reverse Transcription kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The kit includes a poly-A polymerase and a reverse transcriptase. The first step adds a poly-A tail to the 3′-end of the small RNA molecules, while in the second step, the reverse transcriptase converts RNA to cDNA. To quantitate the miRNAs, miScript Primer assays (Qiagen) were used, which include a universal primer specific to the poly-A tail and a miRNA-specific primer. SYBR Green (Qiagen) was used as the florescent molecule. The amplified PCR product had a size of ~80 bp. The small RNA molecule, RNU1A (Qiagen), was used as a control.
Quantitative PCR was performed using LightCycler Instrument 480 (Roche Diagnostics, Mannheim, Germany). The PCR program included a fast start step of 10 min at 95°C followed by 45 cycles of amplification. Each cycle consisted of denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec and elongation at 70°C for 10 sec.
As the reference molecule, miR-205 was used, which showed relatively stable expression [cross-over threshold (Ct) of <30)] throughout all samples and was not significantly affected by the therapy. For quantitation, the comparative ΔCt method was used. Serial dilutions of cDNA were generated using primers for the internal control of miR-205, which exhibited a linear decrease in expression (correlation coefficient=0.99). These dilution series were co-amplified in each PCR session, and the expression values of the internal control and miRNAs from a given sample were derived from the Ct values using the dilution standard. The quantitative experiments were performed twice and the mean values were calculated.
Statistical analyses
The Pearson correlation test was used to evaluate the correlation between the individual miRNA molecules. Mann-Whitney U and Wilcoxon tests were utilized to assess the univariate differences between the pretreatment plasma levels and clinicopathological parameters, or between the changes during neoadjuvant chemotherapy and clinicopathological parameters. Statistical analyses were conducted with SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was used to indicate a statistically significant difference.