Total RNA was extracted using the RNeasy® kit (Qiagen, Courtaboeuf, France) from either 107 cells or 30 mg tumor sample. Before RNA extraction, tumor fragments were crushed with stainless steel beads and homogenized at 30 Hz for 4 min with cold agitation in a MM300 Mixer Mill (Qiagen). Cells or homogenized tissue were lyzed, and RNA purification was performed according to the manufacturer's protocol. A DNase I digestion step was added to each extraction to further decrease genomic DNA contamination. RNA integrity was examined by electrophoresis on a 3% agarose gel, and total RNA concentration was measured by spectrophotometry at 260 nm.Reverse Transcription
RNA (0.5 or 1 µg) was added during the reverse transcription process in order to examine the reproducibility of the experiment. After this step, only 0.5 µg RNA was used to further establish the standard amplification curves and to estimate the range of expression levels in tumor samples. Briefly, RNA was incubated with 20 U avian myeloblastosis virus (AMV) reverse transcriptase (Roche, Meylan, France), 1 mM dNTP, 1.6 µg oligo(dT)15 primer, and 40 U RNase inhibitor in a final volume of 20 µL. Reverse transcription was performed as follows: 25°C for 10 min, 55°C for 1 h (cDNA synthesis and destruction of the RNA portion of the RNA:cDNA hybrids by RNase H activity of recombinant AMV reverse transcriptase), and 5 min at 94°C (enzyme inactivation). Two types of controls were included: (i) the reverse transcription control for each extracted RNA contained all reagents except the AMV reverse transcriptase and (ii) the RNA control was reverse-transcribed without any RNA matrix.
Product DNA of each target gene and reference gene were amplified and purified to establish the standard curve and the calibrator. The DNA precipitation step was started by adding 1/10 volume 2 M sodium acetate to the cDNA reactions. Product DNA was precipitated with 2.5 volumes 100% ethanol (Sigma, St. Louis, MO, USA) at −80°C for 1 h. After centrifugation at 13,000 × g for 45 min at 4°C, the supernatant was discarded, 500 µL 75% ethanol were added and after a second centrifugation at 13,000 × g at 4°C for 30 min, the supernatant was removed. The product DNA pellet was dried and dissolved in 50 µL Tris-HCl.Quantitative PCR of XRCC4, HIF-lα, and HPRT Genes
The primers used for the amplification reaction of each gene are listed in Table 1. They were designed and verified by dedicated software (Amplify 1.2), and primer sequences were analyzed by BLASTn for their specificity. To exclude contamination by residual genomic DNA, the primers were always chosen spanning two different exons. Real-time PCR amplification was performed on a LightCycler (Version 3.5; Roche). Each reverse transcription product and the purified cDNA were used for PCRs. The reverse transcription products from tumor samples were diluted to a concentration such that the CT value of the diluted solution were well within the range of detection capacity of the real-time PCR apparatus that was determined by a standard curve. For the reaction, 2 µL pure or diluted reversetranscribed products or the purified cDNA products were incubated with 4 mM MgCl2, 2 µL ready-to-use SYBR® Green I Master mix (LightCycler FastStart DNA Master SYBR Green I; Roche), and 0.5 µM forward and reverse primers in a final volume of 20 µL. The conditions for PCR and melting curves performed after amplification were tested and optimized. The amplification product was then submitted to specific restriction endonuclease digestion (PvuII for XRCC4, EcoRI for HIF-1α, and MboI for HPRT) and polyacrylamide gel electrophoresis (PAGE).
Gene Expression Quantification
The real-time PCR machine (LightCycler) measured the fluorescence of each sample in every cycle at the end of the elongation step. After amplification, all fluorescence data sets were then exported by the LightCycler software in the form of text files for SCF analysis. SigmaPlot® (Version 9; Systat Software, Richmond, CA, USA) was used to fit fluorescence readings with a nonlinear regression function. Real-time PCR can be precisely simulated using the four-parametric sigmoidal function:
in which C is the cycle number, Fc is the reaction fluorescence at cycle C, Fmax is the maximal fluorescence during the reaction, C1/2 is the cycle at which fluorescence reaches half of Fmax, k is related to the slope of the sigmoid curve (at C = C1/2, the slope = Fmax/4k), and Fb is the background reaction fluorescence. Because fluorescence intensity increase is proportional to the product concentration increase, the Fc calculated from Equation