age: 73 gender: w disease: Squamous Cell Carcinoma received platinum-based therapy with progression: no
Extracted molecule
total RNA
Extraction protocol
RNA of LCM-tumor cells was extracted using the RNeasy Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and solutions are part of the kit and were applied according to the manufacturer’s instructions. In brief: LCM tumor cells suspended in 350µl lysis buffer were incubated with 350µl 70% ethanol. 700µl suspension was put on RNeasy MinElute- column and spun down for one minute with 10,000 rpm. DNase- digestion (10µl DNase+70µl RDD buffer-> 80µl) for 15 at 30°C. 2 washing steps with 350µl RW1- buffer and 500µl RPE- buffer. 500µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2 minutes with 10,000 rpm. Removal of ethanol: column was spun down for 5 minutes with 30,000 rpm. Elution of RNA with 14µl of RNAse free water. Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab Biotechnologie GmbH, Erlangen). Purified RNA was stored at -80°C.
Label
Biotin/Streptavidin-PE
Label protocol
Amplification, reverse transcription and labelling of the sample. For amplification of micro-dissected RNA, the MessageAmp™ aRNA Kit (Ambion, Austin, TX, USA) was used. Between 200-500ng of isolated RNA were transcribed into cDNA. First strand cDNA- Synthesis: 11µl (RNA or RNA and dH2O) Sample 1µl Oligo(dT)Primer 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit (stated below). Reaction kit: 2µl 10x First Strand Buffer 1µl Ribonuclease Inhibitor 4µl dNTP Mix Adding 1µl Reverse transcriptase and 2 h incubation at 42°C Second strand cDNA- Synthese: 63µl Nuclease-free Water 10µl 10x Second Strand Buffer 4µl dNTP Mix 1µl RNase H For second strand sythesis 2µl der DNA- Polymerase are added followed by a 2 h incubation at 42°C. Purification of cDNA: Final elution volume = 16 µl and storage for 12 h at -20°C Labelling with in vitro transcription using biotin labelled nucleotides: Labeling mix: 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit, Enzo Life Sciences, Farmingdale) 4µl T7 10x reaction buffer 12µl RNase free water The labelling mix is incubated with the sample and 4µl of the enzyme mix is added followed by an incubation period of 5 hours at 37°C. The amplified RNA is purified and is eluted in a final volume of 96 µl. Quality control of aRNA: The concentration and purity of aRNA is measured using the NanoDrop ND-1000 (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm The OD 260/OD 280 aRNA was between 1.7 and 2. To exclude the possibility of degradation and/or contamination RNA was analyzed again using a Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto CA). Fragmentation: For optimal hybridization with the complementary oligonucleodid sequences, a fragmentation of the amplified RNA was performed. 24µl of the sample were added to 6µl of fragmentation buffer (200mM TRIS-Acetat, ph 8.2, 500mM KOAc, 150mM MgOAc, Affymetrix) and incubated at 94°C for 35 minutes.
Hybridization protocol
Arrays were prehybridizised for 10 minutes using 140µl MES- hybridization buffer (70,4g MES free acid monohydrate, 193.3g MES Sodium Salt, 800ml accugen water, pH 6.5 – 6.7) and 140µl aqua dest. at 45°C and 60rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix). After removal of the pre-hybridizing solution, the array is loaded with 250 µl of the hybridizing solution (30µl fragmented aRNA, 5µl control-oligo B2, 15µl 20x Eukaryotic hybridizing control, 3µl salmon sperm-DNA, 3µl azetylic BSA, 150µl 2x MES- Hybridizing buffer and 94µl aqua dest.). Following 16-hour incubation at 45°C with permanent rotation, the hybridizing solution is removed. The array is loaded with 280µl of washing buffer A (300ml 20x SSPE, 1ml 10% Tween 20, 698ml accugen water) and a maximum of 4 hybridized arrays are put into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B (83.3ml 12x MES, 5.2ml 5M NaCl, 1ml 10% Tween 20, 910.5ml accugen water) using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus Oligonukleotid- Arrays = Midi-euk2v3). During washing procedure the staining solution is prepared: Staining solution: 600µl 2x Staining Buffer 540µl dH2O 48µl BSA 12µl Streptavidine- Phycoerythrin- Conjugate Array is incubated with 600µl of the staining solution after washing for 40 minutes followed by incubation with the antibody solution for 10 minutes: Antibody solution: 300µl 2x Staining Buffer 266 4µl dH2O 24µl BSA 6µl Goat IgG 3.6µl biotinylated Anti- Streptavidin- AB After removal of the antibody solution 600µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
Scan protocol
The scanning of the array is performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome phycoerythrin at a wave length of 488nm with an emission at 570nm.
Description
Laser captured primary lung cancer cells
Data processing
Using the affy package of functions of statistical scripting language 'R' integrated into the Bioconductor project data from cel files of each sample were normalized by variance stabilizing transformations (VSN) Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics 2002;18:96-104.
