tumor tissue samples from surgically treated patients were collected intraoperatively, snap-frozen and stored at -80 C. All samples underwent a careful analysis of the histopathology, estimation of tumor and stromal content and subsequent macro-dissection of the tumor regions before enrollment into the microarray experiments.
Extracted molecule
total RNA
Extraction protocol
total RNA was extracted using the RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturers instructions. Reverse transcription was performed with 2 ug total RNA per reaction using RevertAid First Strand cDnull Synthesis Kit (Fermentas, Burlington, ON, Canada) according to the manufacturers instructions.
Label
FAM dye
Label protocol
N/A
Hybridization protocol
N/A
Scan protocol
N/A
Description
gene-specific primer and probes (TaqMan Assays, Applied Biosystems, Weiterstadt, Germany) were used in combination with QPCR Master Mix Reagent (Abgene, Epsom, UK) in 384-well format for the ABI Prism 7900HT Sequence Detection System (AB) . The input amount of cDnull in one single PCR reaction corresponded to 10 ng total RNA. PCR-amplification started with the activation of the Taq polymerase at 50 C for 2 min and 95 C for 15 min, followed by 40 cycles of 15 sec at 95 C (denaturation) and 60 sec at 60 C (annealing and elongation).
Data processing
The accuracy of measurements for all samples was optimized by using technical triplicates. Statistical analysis was performed calculating the median Ct value (number of PCR cycles up to a constant fluorescent signal threshold) across the triplicates of every sample within the logarithmic PCR cycle phase and the related median absolute deviation (MAD) respectively. Samples with missing values (Ct value > 40 cycles) were not included in the advanced statistical analysis. To evaluate differences in gene expression, a relative quantification method based on the delta delta Ct-method was used [Pfaffl MW, Nucleic Acids Res, 2001]. Therefore, the gene expression value Ct of a sample for every experimental gene (Gx) were normalized (delta Ct = CtGx-CtGh) using the mean (Gh) of two housekeeping genes (Esterase D and Dnull directed RNA polymerase II polypeptide A), and compared to a reference value (median of all delta Ct values from normal bronchial samples or the matched normal sample) obtaining the normalized, relative value (delta delta Ct) or expression level (2^-delta delta Ct). Additionally, for each expression level an error is calculated based on the MAD divided by the square root of the triplicate Ct values of a sample.
Submission date
Mar 25, 2008
Last update date
Apr 21, 2009
Contact name
Ruprecht Kuner
Organization name
German Cancer Research Center and National Center of Tumor Diseases
