mRNA was isolated with an mRNA mini kit (Oligotex Direct; Qiagen). First-strand cDNA synthesis and amplification were performed with a reagent kit (RT
2 First Strand Kit; SA Biosciences, Frederick, MD). Primers used in the real-time RT-PCR were specific for human hypoxanthine phosphoribosyl transferase (
hHPRT) sense primer 5′-GACCAGTCAACAGGGGACAT-3′, antisense primer 5′-AAGCAGATGGCCACAGAACT-3′, mouse glyceraldehyde 3-phosphate dehydrogenase (
GAPDH) sense 5′-ACTCACGGCAAATTCAACGGC-3′, and antisense 5′-ATCACAAACATGGGGGCATCG-3′. Real-time RT-PCR amplifications were performed on a real-time PCR detection system (iCycler IQ; Bio-Rad, Hercules, CA) using a standard protocol (95°C, 10 minutes, 40 cycles; 95°C, 15 seconds, 60°C, 1 minute). Each analysis was performed in triplicate, in a 96-well PCR plate sealed with an optical adhesive cover. Nontemplate controls (water) were included in each assay. A mix for real-time PCR applications (IQ SYBR Green SuperMix 2× Reaction System kit; Bio-Rad) was used, with a final volume of 25 μL containing fluorescein for dynamic well factor collection on a detection system (iCycler iQ; Bio-Rad). For each analysis, 5 μL diluted DNA was used. After several trials, the primer concentrations were fixed at 0.2 μM. The Δ
C t data were collected automatically. The average Δ
C t of each group was calculated with the following formula: Δ
C t = average hHPRT gene
C t − average mGAPDH gene
C t. ΔΔ
C t was calculated by ΔΔ
C t = Δ
C t of control SiRNA group − Δ
C t CXCR4 SiRNA group. The fold-change for
hHPRT expression level was calculated using 2
−ΔΔC1 .
17,18