Sample code: T19 Cell type: tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue Gender: male Age at time of surgery: 71 Smoking status: current Packyears: 51 Type of Surgery: lobectomy Tumor type: squamous cell lung carcinoma Location tumor: left upper lobe TNM stage: T2N0M0 Tumor stage: IB Dead/alive at last follow-up: dead Survival as from surgery: 13 months Metastases: distant Time to distant metastasis: 13 months COPD: no COPD FEV1: 108 % FEV1/FVC: 79 % GOLD stage: not applicable
Extracted molecule
total RNA
Extraction protocol
LASER DISSECTION MICROSCOPY - Frozen squamous cell lung carcinoma tissue sections of 8μm were used for laser dissection microscopy (LDM) - Only vital tumor cells without apparent admixture of inflammatory cells through the tumor fields were isolated using laser dissection microscopy - An area of approximately 20x106 μm2 was microdissected from the section using P.A.L.M. Microlaser Technology system (P.A.L.M., Bernried, Germany). - Laser microdissected cells were collected in lysis buffer (Macherey-Nagel) RNA ISOLATION Total RNA was isolated and purified from the microdissected cells with a Nucleospin RNA II kit (Macherey-Nagel). The quantity of DNA-free total RNA was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA quality was assessed by the presence and ratio of 18S and 28S rRNA bands combined with a low baseline, monitored with RNA 6000 PicoChip (Agilent, Palo Alto, CA) on the 2100 bioanalyzer (Agilent). RNA AMPLIFICATION For each sample, 150 ng total RNA was amplified using the MessageAmp II kit (Ambion, Austin, USA). Briefly, 150 ng total RNA was mixed with 1 ul of T7 Oligo(dT) primer in a total volume of 12 ul. Primer and template were denatured by incubating at 70C for 10 minutes and annealed by putting the reaction tubes on ice. The First Strand Reaction was performed by adding 8 µl Reverse Transcription Master Mix (containing 10x First Strand buffer, Ribonuclease Inhibitor, dNTP Mix and Reverse Transcriptase) and incubating at 42C for 2 hours. Second Strand cDNA Synthesis was done by adding 63 µl Nuclease-Free Water, 10 µl 10x Second Strand Buffer, 4µl dNTP Mix, 2µl DNA Polymerase and 1 µl RNase H and incubating at 16C for 2 hours. cDNA purification was done according manufacturers protocol (Ambion). In vitro transcription was initiated by addition of 2 µl aaUTP Solution (50mM), 12 µl ATP, CTP, GTP mix (25mM), 3 µl UTP Solution (50mM), 4 µl T7 10x Reaction Buffer and 4 µl T7 Enzyme Mix and incubated at 37C for 9 hours. Machery-Nagel RNA Clean up mini spin columns are used for purification of the cRNA.
Label
Cy3 and Cy5
Label protocol
ULS LABELING 1. Take 1- 2 μg of aRNA. Ensure that the final concentration in the labeling reaction is above 50 ng/μL. Optimal modification degrees of the labeled material are not achieved if the final concentration of aRNA in the labeling mixture is below 50 ng/μL) 2. Add 1 μL of Cy5-ULS or Cy3-ULS per 1 μg aRNA (Always keep the ratio of μg of aRNA to μL ULS 1:1 when increasing or decreasing the amount to be labeled). 3. Add 1/10 volume of 10 x Labeling solution 4. Adjust with RNase-free water to final volume and mix by pipetting 5. Label sample by incubation for 15 minutes at 85°C 6. Place samples on ice, spin down to collect contents of tube before proceeding with purification using the KREApure columns Dye Removal using KREApure Columns (Removal of free ULS label using KREApure columns (20800 x g is equivalent to 14,000 rpm on eppendorf 5417C)): 1. Resuspend column material by vortexing 2. Loosen cap ¼ turn and snap off the bottom closure 3. Place the column in a 2 mL collection tube 4. Pre-spin the column for 1 minute at 20800 x g 5. Discard flow through and column cap, but re-use collection tube 6. Add 300 μL ultrapure water to the column and centrifuge for 1 min at 20,800g 7. Discard collection tube and flow-through 8. Place column in a new RNase free 1.5 mL microcentrifuge tube (not provided) 9. Add ULS-labeled aRNA onto column bed 10. Centrifuge column for 1 minute at 20800 x g 11. Flow through contains the purified labeled aRNA
Hybridization protocol
Hybridization was performed with 750 ng of each labeled target together with fragmentation- and hybridization buffer at 60C for 17 hours onto Agilent Whole Human Genome oligoarrays following manufacturer’s protocol.
Scan protocol
The microarray slides were washed following the user manual instructions and scanned using the Agilent dual laser DNA microarray scanner. The microarray data were normalized using the Agilent feature extraction software v. 8.5 (Agilent Technologies, Palo Alto, CA) with regards to no background correction.
Description
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Data processing
The gene expression data were log transformed and quantile normalized (Cy3 and CY5 separate). For both Cy3 and Cy5, a filter was applied that excluded 50% of the probes with the lowest expression levels and 25% of the probes with the lowest expression range. Probes that had passed only one Cy-dye filter were excluded. The average of the normalized Cy3 and Cy5 data were used for further analysis.
