Patients and samples
Kidney tissue samples derived from 25 adult patients with RCC (21 male, four female, mean age 62 years, range: 45 to 92 years) undergoing radical nephrectomy at the Department of Urology of the University Hospital Charité between September 2003 and January 2006. The use of the tissue material for research was approved by the Medical Ethical Committee of the Charité Hospital (Chairman: Prof. R. Uebelhack, University Hospital Charité, Berlin, Germany; protocol "Detection of metalloproteinases in patients with genitourinary cancer"; July 16, 2002). Matched malignant and non-malignant specimens from the same kidney were collected immediately after surgery in tubes with RNAlater® Stabilization Reagent (Qiagen, Hilden, Germany), stored at 4°C overnight, and then put in long-time storage at -80°C until RNA isolation.
Tumour stage and classification were established according to the 2002 TNM System and the 2004 WHO Classification [12,45]. All tumours were clear cell carcinoma (ccRCC). Eleven of the 25 tumours studied were classified stage pT1, two tumours pT2, and 12 tumours pT3. The histological grading was once G1, 23 times G2, and once G3. None of the patients had metastases (M0 and pN0).
RNA isolation and characterization
Total RNA was isolated from about 50 mg of preserved kidney tissue samples cut into small pieces and homogenized in 350 μl RNA lysis/binding buffer including 1% beta-mercaptoethanol. The RNeasy Mini Kit (Qiagen) was used for RNA isolation according to the manufacturer's instructions. An additional digestion step on the RNA binding silica gel membrane of the spin column was performed with DNase I. The RNA yield and the ratio of absorbance at 260 nm to 280 nm (A260/A280 ratio) were measured with the NanoDrop®ND-1000 Spectrophotometer (NanoDrop Technologies, Montchanin, DE, USA).
The integrity of isolated total RNA was assessed with the RNA 6000 Nano LabChip® kit using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Agilent 2100 Expert software was used to generate a so-called RNA Integrity Number (RIN) as criterion of the RNA quality for downstream experiments. The RIN values are scaled from number 1 (RNA completely degraded) to 10 (intact RNA) [27,35,36].
First strand cDNA synthesis
One μg RNA was reversely transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Penzberg, Germany) with random hexamer priming method according to the manufacturer's recommendations. This kit was selected as result of previous comparative studies between various cDNA synthesis kits. That kit offers a fast, complete, and high-yield cDNA synthesis [46]. Briefly, RNA samples and random primers were mixed and denatured for 10 min at 65°C. Thereafter, tubes were immediately placed on ice. The first strand cDNA synthesis was started after adding transcription mixture at 25°C for 10 min (random primer annealing) following 30 min at 55°C for reverse transcriptase reaction. Finally, the enzymes were inactivated at 85°C for 5 min. Each RNA sample was controlled for genomic contamination without reverse transcriptase addition into cDNA synthesis mixture. cDNA samples were stored at -20°C and diluted 1:5 with RNase-free water for use as template in real-time PCR analysis.
Real-time RT-PCR
Real-time PCR was performed with the LightCycler instrument (Roche) by using different measurement and detection modes. The primer and probe sequences applied for the cDNA amplification of the ten candidate reference genes and one target gene are given in Table 2. The primer/probes sequences and PCR run conditions for the detection of ACTB, ALAS1, GAPDH, HMBS, HPRT1, SDH, and TBP were used as previously described [9]. For PPIA, RPLPO, and TUBB, real-time PCR pre-designed assays (Qiagen) based on detection with SYBR Green I (SGI) fluorescence dye were used. cDNA amplification for these three genes were performed with the QuantiTect SYBR Green Master Mix (Qiagen) that contained HotStar Taq DNA polymerase, dNTPs, MgCl2 and a special buffer system with SGI dye. The final PCR reaction mix included 0.5 μmol/l of specific primer assay mixtures, 1 μl diluted cDNA, DNase-free water and 2 μl QuantiTect SGI PCR Master mix at a final volume of 10 μl. The cycle conditions were set for PPIA, RPLPO and TUBB as follows: Taq DNA polymerase activation at 95°C for 15 min with a ramping rate of 20°C/s, start of each amplification cycle with a denaturation step at 94°C for 15 s, primer annealing for PPIA at 55°C for 20 s and for RPLPO and TUBB at 57°C for 20 s, primer extension at 72°C for 20 s. The ramping rate was 2°C/s for all 38 or 45 PCR cycles.
The target gene ADAM9 cDNA amplification was performed with the ready-to-use LightCycler® FastStart DNA MasterPLUS HybProbe (Roche). The final reaction concentrations of both primers were 0.5 μmol/l and the donor/acceptor probe concentrations were 0.2 μmol/l each. The PCR setup was: activation of FastStart Taq DNA Polymerase at 95°C for 15 min, followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 62°C for 30 s and elongation at 72°C for 30 s. The temperature transition rate was 20°C/s. The final PCR volume of 20 μl included 1 μl of 1:5 diluted cDNA.
In dependence on gene-specific primer/probe sets for real-time PCRs, the amplification rate detected as fluorescence accumulation was gained by using probes after annealing phase and by using SGI after elongation step. To evaluate fluorescence data the method of Second Derivative Maximum of the LightCycler Software 3.5 (Roche) was utilized. Crossing point (Cp) values as well as quantities were applied for comparison of gene expressions in paired samples. For the quantification of each gene a pooled cDNA sample with a high expression value was stepwise diluted and amplified. The undiluted sample was set as one. The resulting standard curve was given in arbitrary units. In each PCR run a diluted secondary standard was used as calibrator and another cDNA dilution was used as run-to-run precision control. Prior to this, the control cDNA pool was adjusted in the range of sample expression level. In the same procedure the PCR efficiencies were calculated (Table 2).
Each PCR run included a no template control with water instead of cDNA. Duplicate measurements were performed and mean values were used for all further calculations. To minimize the analytical variation paired malignant and non-malignant samples were always analysed in the same PCR run.
Data analysis
Statistical analyses were performed with GraphPad Prism for Windows, version 4.03 (GraphPad Software, San Diego, CA, USA). The distribution fitting procedure according to the D'Agostino & Pearson omnibus normality test was performed and non-parametric tests (Wilcoxon test for paired samples; Mann-Whitney test) were applied. Correlations were characterized by the Spearman's rank correlation coefficient rs. P
To characterize the expression stability of the candidate reference genes, the programs geNorm, version 3.4 [4,28] and NormFinder [29,30] were applied. geNorm processes gene expression data as concentration values, taking into account the PCR efficiencies of investigated genes (Table 2) [4]. The most stable genes are stepwise selected from the investigated gene panel and a common Normalization Factor (NF) can be calculated for the genes selected for the normalization procedure.
The program NormFinder is a free Add-in for Microsoft Excel [29,30]. NormFinder processes data in linear scale. On the basis of a given "group identifier", the program can discriminate between different groups, e.g. malignant and non-malignant samples. The program algorithm implies the estimation of intra- and intergroup variation and combines both results in a stability value for each investigated gene. The candidate gene with the lowest stability value is the most stable gene within the groups studied. The best combination of two genes is also indicated.
Answers to all your Biology Questions
