diagnosis: squamous cell lung carcinoma location: Tumor Center
Treatment protocol
RNA samples from each patient were distinguished according to i) inner tumor, ii) tumor invasion front, iii) adjacent lung front, and iv) normal lung
Growth protocol
Primary squamous cell lung carcinoma samples were obtained from the Thorax Clinic, University of Heidelberg, Germany.
Extracted molecule
total RNA
Extraction protocol
For RNA isolation, microdissected tissues from consecutive cryosections were pooled into a final volume of 350 μL QIAzol Lysis Reagent (Qiagen, Hilden, Germany). Total RNA extraction was carried out using a miRNEasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and subsequently examined for integrity using a 2100 Bioanalyzer (Agilent, Santa Clara, CA).
Label
Cy5,Cy3
Label protocol
For amplification and labeling 25 ng of total RNA from each area or reference mRNA (Universal Human Reference RNA, Agilent) were subjected to two rounds of linear amplification using the in-vitro transcription-based TAcKLE protocol (Schlingemann J, Thuerigen O, et al., 2005). Subsequent generation of cDNA yielded separately labeled Cy3 and Cy5 tumor- and reference samples, respectively. (21).
RNA samples from each patient were distinguished according to i) inner tumor, ii) tumor invasion front, iii) adjacent lung front, and iv) normal lung
Growth protocol
Primary squamous cell lung carcinoma samples were obtained from the Thorax Clinic, University of Heidelberg, Germany.
Extracted molecule
total RNA
Extraction protocol
For RNA isolation, microdissected tissues from consecutive cryosections were pooled into a final volume of 350 μL QIAzol Lysis Reagent (Qiagen, Hilden, Germany). Total RNA extraction was carried out using a miRNEasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and subsequently examined for integrity using a 2100 Bioanalyzer (Agilent, Santa Clara, CA).
Label
Cy3,Cy5
Label protocol
For amplification and labeling 25 ng of total RNA from each area or reference mRNA (Universal Human Reference RNA, Agilent) were subjected to two rounds of linear amplification using the in-vitro transcription-based TAcKLE protocol (Schlingemann J, Thuerigen O, et al., 2005). Subsequent generation of cDNA yielded separately labeled Cy3 and Cy5 tumor- and reference samples, respectively. (21).
Hybridization protocol
Purified and dye-labeled sample and reference cDNA was combined for two-color hybridizations with 520 µl hybridization buffer (4x SSC, 50% formamide, 2% SDS), agitated for 30 min at 60 °C, heated for 10 min at 70 °C on a thermo mixer and subsequently applied to preheated (60 °C) gasket slides (Agilent Technologies, Santa Clara, USA). Microarrays were place onto loaded gasket slides, assembled in SureHyb hybridization chambers (Agilent) and incubated for 23 hours at 42 °C and a rotation speed of 4 rpm in an Agilent DNA Microarray Hybridization Oven. Hybridized arrays were washed at 35 °C with (i) 0.5x SSC, 0.1% SDS for 4 minutes; (ii) 0.05x SSC, 0.1% SDS for 4 minutes; (iii) 0.05x SSC for 2 minutes; (iv) 0.05x SSC, 0.05% Tween-20 for 30 seconds. Immediately thereafter, slides were dried by centrifugation in 50 ml Falcon tubes at 2500 rpm for 5 minutes. All hybridization experiments were repeated with inversely labeled sample and reference.
Scan protocol
Microarray read out was accomplished in a two-color Agilent Scanner G25505B with 5 µm resolution and automatically adjusting PMT voltages according to manufacturer’s specification. Recorded images were analyzed using GenePix Pro 6.0 software (Axon).
Description
squamous cell lung carcinoma _Tumor Center_2502
Data processing
Following image extraction, low quality spots were filtered and the background intensities were corrected by normexp with offset 50 (Ritchie, ME, Silver, J, et al., 2007) and within array robustspline normalization was applied. Quantile normalization was applied between arrays for further normalization. Annotation of oligonucleotide probes present on the gene expression array was retrieved by alignment to EnsEMBL database version 55_37.
