Sample code: T18 Tissue type: Human lung tumor (frozen) Gender: Male Age at time of surgery: 70 Packyears: 36 Tumor type: Squamous cell lung carcinoma (SCC) Type of Surgery: Bilobectomy Location tumor: Right upper lobe TNM stage: T2N0M0 Tumor stage: IB Metastases: distant Time to distant metastases as from surgery (months) : 9 Location of distant metastasis: left lung Dead/Alive: dead Survival as from SCC resection (months): 23 COPD: COPD FEV1: 84 % FEV1/FVC: 54 % GOLD stage: GOLD I
Extracted molecule
genomic DNA
Extraction protocol
LASER DISSECTION MICROSCOPY - Frozen tumor 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 25x106 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 20 μl SE buffer DNA ISOLATION USING A STANDARD SALT-CHLOROFORM EXTRACTION PROTOCOL: - Add 400 μl SE buffer, 20 μl 10% SDS and 2 μl Proteinase K (20 mg/ml) - Incubate the solution overnight at 55°C in the incubator; also incubate the saturated NaCl solution (6M) overnight at 55°C - Add 134 μl preheated saturated NaCl solution to the solution - Add 1 volume (556 μl) chloroform - Mix 1 hour on a top-over-top rotor - Centrifuge for 2 minutes, 15000 rpm, 4°C - Transfer upper phase to a clean tube - Add 1 volume isopropanol and mix carefully - Centrifuge for 2 minutes, 15000 rpm, 4°C - Wash pellet/cloud with 200 μl 80% ethanol - Centrifuge for 2 minutes, 15000 rpm, 4°C - Pipette off all the ethanol, shortly centrifuge and pipette off again - Dry pellet on air for 5-10 minutes - Dissolve pellet in 20 μl TE-4 - The DNA concentration was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) For each sample, 100 ng genomic DNA was amplified using the BioScore kit (Enzo Life Sciences, Farmingdale, NY): AMPLIFYING THE GENOMIC DNA In order to establish the lower and upper limits of the amplification reaction, a negative control reaction (no template DNA) and a positive control reaction (100 ng of Excellent high quality, non-FFPE genomic DNA) should be included in the protocol. We recommend amplifying the same reference DNA that will be labeled for array analysis (e.g., pooled normal male or pooled normal female from Promega). 1. Combine 100 ng (up to 19 μl) of genomic DNA (FFPE-extracted or non-FFPE) with 20 μl Primers (P) and add enough Nuclease-free Water (W) to bring the reaction mixture to 39 μl. Mix by gently flicking tube, centrifuge briefly. 2. Heat at 99oC for 10 minutes. Place tubes immediately on ice for 5 minutes. Centrifuge briefly and return to ice. 3. While on ice, add 10 μl Nucleotide Mix (N) and 1 μl Enzyme (E). Mix by gently flicking the tube. Centrifuge briefly and incubate at 37oC for 1 hour. 4. Add 5 μl of Stop Buffer (S). Mix by gently flicking the tube. Centrifuge briefly and keep at room temperature. PURIFYING THE AMPLIFIED DNA 1. Use the QIAquick PCR Purification Kit. Add 300 μl of PB Buffer (≥ 5 volumes) to each tube. Mix thoroughly by repeated inversion and centrifuge briefly. Add each sample to a separate QIAquick column. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate. 2. Add 750 μl of Buffer PE. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate 3. Add 750 μl of Buffer PE. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate. Transfer the QIAquick column to a new collection tube. Centrifuge again at 13,000 RPM (16,000 x g) for 1 minute. Transfer the QIAquick column to a new 1.5 ml microfuge tube. 4. Add 25 μl of Buffer EB (preheated to 37oC). Incubate at RT for 1 minute. Elute the amplified DNA by centrifugation at 13,000 RPM (16,000 x g) for 1 minute. Add an additional 25 μl of Buffer EB (preheated to 37oC). Incubate at RT for 1 minute. Centrifuge at 13,000 RPM (16,000 x g). 5. Measure concentration of the amplified DNA by spectrophotometric analysis and calculate yield. 6. Store amplified products at 4°C for short-term storage (< 1 week) and at –20oC for long-term storage. INTERPRETING THE RESULTS OF THE AMPLIFICATION REACTION The table below indicates the DNA yields expected from a 1 hour amplification of 100 ng of input DNA. These values are not applicable for reactions longer than 1 hour or with inputs greater than or less than 100 ng. Samples with yields in the Poor range are unlikely to yield informative array results and should not be used. DNA Quality Total Yield (ug) No input <0.5 Poor <1 Intermediate 1-3 Good 3-10 Excellent >10 The exact yields used for scoring DNAs may vary slightly depending on the user’s labeling conditions and array platform. LABELING FOR ARRAY ASSAYS 1. Direct Labeling of Unamplified DNA - 0.85 μg of Good genomic DNA can be used for direct labeling and is likely to yield excellent array results. - >0.85 μg of Intermediate genomic DNA should be used for direct labeling (e.g., 1.7 μg). - Poor input DNA should not labeled. 2. Labeling of Amplified DNA - 0.85 μg of amplified DNA from 100 ng of genomic sample DNAs scored as Excellent or Good can be labeled for successful array analysis. - 0.85 μg of amplified DNA from from 100 ng of genomic sample DNAs scored as Intermediate can be labeled for array analysis. These arrays, however, are likely to have higher noise levels than arrays using unamplified DNA. Only samples scored for Excellent or Good were used for further processing.
Label
Cy5
Label protocol
The random primed labelling procedure is carried out with components from the BioPrime DNA Labelling System (Invitrogen). - Mix 600 ng of genomic DNA with 20 µl of 2.5x random primer solution in a 200 µl vial and make up the volume to 42 µl with MilliQ-water - Denature the DNA by heating the mixture at 99oC for 10 min. in the MJ-Cycler. Briefly centrifuge and place on ice for 10 min. - Add: 5 µl10xdNTP mixture, 2 µl Cyanine 3-dUTP or Cyanine 5-dUTP, 1 µl Klenow DNA polymerase - Mix well and incubate the vials for 16 hours in the MJ-cycler at 37 oC. - Pre-spin a MicroSpin G50 Column at 750g in the Eppendorf centrifuge according to the users manual of the columns. - Pool the labelled test and reference DNA-samples and amply this mixture on top of the pre-spinned column. Centrifugate the column for 2 min. at 750g and collect the purified labeled DNA in a fresh tube. - Apply the collected eluate to a Microcon YM-30 filter and centrifugate at 14000 rpm in the eppendorf centrifuge, until the volume is decreased to approximately 5 µl. (app. 5 min.) - Apply 50 µl of the prepared hybridisation mix to the filter and incubate for 10 min. - Collect the labelled DNA in hybridisation-mixture by inverting the filter into a fresh 1.5ml tube and spin for 2 min. at 9000 rpm. The eluate should show a purple colour.
Whole blood was collected in disodium EDTA containers and stored at -20ºC and a number of samples were also stored at -80ºC. To facilitate haemolysis of RBCs it is recommended to store a fresh sample for a few hours in a freezer as freezing destroys the red cells. After thawing, 3mls of whole blood are transferred to a sterile conical centrifuge tube (15ml volume) to which 9mls of 1 x erythrocyte lysing buffer (0.155M NH4Cl; 10mM KHCO3; 0.1mM Na2 EDTA; pH 7.4) must be added. The solution is left for 10 minutes at RT with occasional mixing by inversion followed by centrifugation for 5 minutes at 4000 rpm. After centrifugation the supernatant is discarded and a white pellet will be observed at the bottom of the tube. This pellet must be washed for at least 3 times by adding 3mls of erythrocyte lysing buffer and repeating steps 3 and 4. It is important to breakdown the pellet and rinse it well in erythrocyte lysing buffer in order to clean the white blood cells from remaining heme. 1.5mls of SE buffer (75mM NaCl; 25mM Na2 EDTA; pH 8.0) containing 100mg/ml of Proteinase K and 1% sodium dodecyl sulphate (SDS w/v), are added to the pellet. The tubes are then incubated at a temperature of 37-55ºC (optimal temp for Proteinase K activity) overnight in a water bath or incubator. During this step the white blood cells' membranes are denatured and DNA goes out in solution. After the incubation, 1.5mls of SE buffer together with 750ml of 6M (saturated) NaCl are added to each tube, followed by the addition of 3.75mls chloroform. The tubes are mixed vigorously (on a vortex) for about 20 sec with occasional mixing for at least 30min. Alternatively you can leave the tubes on a rotator for 1 hour. The emulsion will then be centrifuged for 10 minutes at 2000 rpm with minimal breaking force. After centrifugation 2 phases are observed and care must be taken not to disturb the interphase. During this step DNA is extracted into the supernatant and proteins separated into the lower phase. The upper aqueous phase (containing the DNA) is transferred into a clean and sterile conical centrifuge tube using a sterile Pasteur pipette, followed by the addition of an equal volume of isopropanol. DNA will be precipitated by gentle swirling of the tube and is observed visually as a white thread like strand. Using a sterile spatula or loop transfer the DNA strand into a sterile microcentrifuge tube containing 1ml of 75% ethanol. The DNA is then washed by inversion to clean it from any remaining salts and the tube centrifuged at 11000g for 4 minutes. The supernatant is discarded taking care not to discard the pellet. Repeat this step once more. After discarding the supernatant the pellet is dried from excess ethanol either by using a vacuum centrifuge or by leaving the tubes open and inverted in an oven at around 50 - 65ºC for an hour. The dried pellet is resuspended in TE buffer (1M Tris-HCl; 0.5M EDTA; pH 8.0) and left overnight on a rotator. DNA concentration is determined either by agarose gel electrophoresis or spectrophotometry and adjusted to the desired concentration by adding more TE buffer. It is important to note that before adjusting and reading DNA concentrations one must obtain a homogeneous sample of DNA which is not quite easy acquired since DNA is very viscous. To adjust to lower concentration one must used other quantitation methods such as Picogreen (Molecular Probes) using spectrofluorometry as spectrophotometry is not accurate and sensitive at very low concentrations. For each sample, 100 ng genomic DNA was amplified using the BioScore kit (Enzo Life Sciences, Farmingdale, NY): AMPLIFYING THE GENOMIC DNA In order to establish the lower and upper limits of the amplification reaction, a negative control reaction (no template DNA) and a positive control reaction (100 ng of Excellent high quality, non-FFPE genomic DNA) should be included in the protocol. We recommend amplifying the same reference DNA that will be labeled for array analysis (e.g., pooled normal male or pooled normal female from Promega). 1. Combine 100 ng (up to 19 μl) of genomic DNA (FFPE-extracted or non-FFPE) with 20 μl Primers (P) and add enough Nuclease-free Water (W) to bring the reaction mixture to 39 μl. Mix by gently flicking tube, centrifuge briefly. 2. Heat at 99oC for 10 minutes. Place tubes immediately on ice for 5 minutes. Centrifuge briefly and return to ice. 3. While on ice, add 10 μl Nucleotide Mix (N) and 1 μl Enzyme (E). Mix by gently flicking the tube. Centrifuge briefly and incubate at 37oC for 1 hour. 4. Add 5 μl of Stop Buffer (S). Mix by gently flicking the tube. Centrifuge briefly and keep at room temperature. PURIFYING THE AMPLIFIED DNA 1. Use the QIAquick PCR Purification Kit. Add 300 μl of PB Buffer (≥ 5 volumes) to each tube. Mix thoroughly by repeated inversion and centrifuge briefly. Add each sample to a separate QIAquick column. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate. 2. Add 750 μl of Buffer PE. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate 3. Add 750 μl of Buffer PE. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate. Transfer the QIAquick column to a new collection tube. Centrifuge again at 13,000 RPM (16,000 x g) for 1 minute. Transfer the QIAquick column to a new 1.5 ml microfuge tube. 4. Add 25 μl of Buffer EB (preheated to 37oC). Incubate at RT for 1 minute. Elute the amplified DNA by centrifugation at 13,000 RPM (16,000 x g) for 1 minute. Add an additional 25 μl of Buffer EB (preheated to 37oC). Incubate at RT for 1 minute. Centrifuge at 13,000 RPM (16,000 x g). 5. Measure concentration of the amplified DNA by spectrophotometric analysis and calculate yield. 6. Store amplified products at 4°C for short-term storage (< 1 week) and at –20oC for long-term storage. INTERPRETING THE RESULTS OF THE AMPLIFICATION REACTION The table below indicates the DNA yields expected from a 1 hour amplification of 100 ng of input DNA. These values are not applicable for reactions longer than 1 hour or with inputs greater than or less than 100 ng. Samples with yields in the Poor range are unlikely to yield informative array results and should not be used. DNA Quality Total Yield (ug) No input <0.5 Poor <1 Intermediate 1-3 Good 3-10 Excellent >10 The exact yields used for scoring DNAs may vary slightly depending on the user’s labeling conditions and array platform. LABELING FOR ARRAY ASSAYS 1. Direct Labeling of Unamplified DNA - 0.85 μg of Good genomic DNA can be used for direct labeling and is likely to yield excellent array results. - >0.85 μg of Intermediate genomic DNA should be used for direct labeling (e.g., 1.7 μg). - Poor input DNA should not labeled. 2. Labeling of Amplified DNA - 0.85 μg of amplified DNA from 100 ng of genomic sample DNAs scored as Excellent or Good can be labeled for successful array analysis. - 0.85 μg of amplified DNA from from 100 ng of genomic sample DNAs scored as Intermediate can be labeled for array analysis. These arrays, however, are likely to have higher noise levels than arrays using unamplified DNA. Only samples scored for Excellent or Good were used for further processing.
Label
Cy3
Label protocol
Prior to the hybridisation, the genomic "test" and "reference" DNA are labelled with Cy3 and Cy5 fluorescent dyes respectively. The random primed labelling procedure is carried out with components from the BioPrime DNA Labelling System (Invitrogen). - Mix 600 ng of genomic DNA with 20 µl of 2.5x random primer solution in a 200 µl vial and make up the volume to 42 µl with MilliQ-water - Denature the DNA by heating the mixture at 99oC for 10 min. in the MJ-Cycler. Briefly centrifuge and place on ice for 10 min. - Add: 5 µl10xdNTP mixture, 2 µl Cyanine 3-dUTP or Cyanine 5-dUTP, 1 µl Klenow DNA polymerase - Mix well and incubate the vials for 16 hours in the MJ-cycler at 37 oC. - Pre-spin a MicroSpin G50 Column at 750g in the Eppendorf centrifuge according to the users manual of the columns. - Pool the labelled test and reference DNA-samples and amply this mixture on top of the pre-spinned column. Centrifugate the column for 2 min. at 750g and collect the purified labeled DNA in a fresh tube. - Apply the collected eluate to a Microcon YM-30 filter and centrifugate at 14000 rpm in the eppendorf centrifuge, until the volume is decreased to approximately 5 µl. (app. 5 min.) - Apply 50 µl of the prepared hybridisation mix to the filter and incubate for 10 min. - Collect the labelled DNA in hybridisation-mixture by inverting the filter into a fresh 1.5ml tube and spin for 2 min. at 9000 rpm. The eluate should show a purple colour.
Hybridization protocol
AMOUNT OF HYBRIDISATION-MIX Take note of the area of the array and establish the size of the lifterslip and the amount of necessary (pre-)hybridisation mix: Lifterslip Volume hybridisation-mix 22 x 22 mm 30 µl 22 x 30 mm 50 µl 22 x 60 mm 100 µl This protocol is intended for 50 µl hybridisation mix. If necessary take in to account that the amount of labelled product should be increased or decreased. PREHYBRIDISATION - Block the array slide according to the protocol of the manufacturer. - Prepare the prehybridisation-mix by adding together (per 50 µl prehybrydisation-mix): 50 µl salmon sperm DNA (~10g/l), 2 µl 3M NaAc pH5.3, 100 µl ethanol - Mix the solution and store it in the freezer for 15 min. - Collect the precipitate by centrifugation at 14000 rpm for 10 min. at 4 oC in the eppendorf centrifuge. - Discard the supernatant and wash the pellet with 500 µl 70% ethanol. - Centrifuge for 5 min. at 14000 rpm. - Discard the supernatant, dry the pellet and dissolve it in 50 µl hybridisation mix with 5% dextran sulphate - Apply the necessary amount of prehybridisation-mix to the array, on an heated block and place a fresh ethanol cleaned lifterslip on top of the array, without introducing air-bubbles. - Prehybridise for 2 hours in a waterbath at 65oC using a hybridisation chamber. - Wash off the lifterslip with milliQ-water, and spin dry the array-slide quickly at 800 rpm for 3 min in the Mistral MSE centrifuge. PREPARING THE HYBRIDISATION-MIX (For 50 µl of hybridisation mix) - Add together: 200 µl COT1-DNA (1µg/µl), 10 µl 3M NaAc pH5.3, 470 µl ethanol - Mix the solution and store it in the freezer for 15 min. - Collect the precipitate by centrifugation at 14000 rpm for 10 min. in the eppendorf centrifuge. - Discard the supernatant and wash the pellet with 500 µl 70% ethanol. - Centrifuge for 5 min. at 14000 rpm. - Discard the supernatant, dry the pellet and dissolve it in 50 µl hybridisation mix with 5% dextran sulphate. Perform next steps as much in the dark as possible! LABELING The random primed labelling procedure is carried out with components from the BioPrime DNA Labelling System (Invitrogen). - Mix 600 ng of genomic DNA with 20 µl of 2.5x random primer solution in a 200 µl vial and make up the volume to 42 µl with MilliQ-water - Denature the DNA by heating the mixture at 99oC for 10 min. in the MJ-Cycler. Briefly centrifuge and place on ice for 10 min. - Add: 5 µl10xdNTP mixture, 2 µl Cyanine 3-dUTP or Cyanine 5-dUTP, 1 µl Klenow DNA polymerase - Mix well and incubate the vials for 16 hours in the MJ-cycler at 37 oC. - Pre-spin a MicroSpin G50 Column at 750g in the Eppendorf centrifuge according to the users manual of the columns. - Pool the labelled test and reference DNA-samples and amply this mixture on top of the pre-spinned column. Centrifugate the column for 2 min. at 750g and collect the purified labeled DNA in a fresh tube. - Apply the collected eluate to a Microcon YM-30 filter and centrifugate at 14000 rpm in the eppendorf centrifuge, until the volume is decreased to approximately 5 µl. (app. 5 min.) - Apply 50 µl of the prepared hybridisation mix to the filter and incubate for 10 min. - Collect the labelled DNA in hybridisation-mixture by inverting the filter into a fresh 1.5ml tube and spin for 2 min. at 9000 rpm. The eluate should show a purple colour. HYBRIDISATION - Denature the DNA in the hybridisation mixture at 100 oC for 5 min. - Apply the suitable amount of the denatured mix to the array, on a heated block; place a fresh, ethanol cleaned, lifterslip on top of the array without introducing air bubbles. - Hybridise for ~40 hours in the Gemini Twin shaker waterbath at 65oC using a hybridisation chamber. POST HYBRIDISATION STEPS - Transfer the slide to a washing rack - Wash the slides using the washing solutions described in the Schott Nexterion guide: 10 min. washing solution 1 at 65oC, 5 min. washing solution 2 at 20oC, 5 min. washing solution 3 at 20oC - Spin dry the array-slide quickly at 800 rpm for 3 min in the Mistral MSE centrifuge. - Scan the array using the Agilent scanner with optimal settings according to the manual. INTERNAL CONTROL Use a reference-DNA sample with opposite gender for these hybridisations. Check the altered ratio’s after a calculation procedure. On the array’s control spots are present, derived from Total Human DNA, CoT-DNA and Drosophila DNA. The intensities of these spots were taken into account in the calculation procedure.
Scan protocol
Agilent scanner (resolution 5 um) in combination with Agilent software. Default settings and protocols as supplied by Agilent
The scanned images were processed with Bluefuse v3.5 (BlueGnomeLtd, Cambridge, UK). A block-median normalization excluding controls was applied to the 2log-transformed Cy3/Cy5 ratios. Subsequent exclusion criteria were: confidence <0.3, quality <1 and SD of replicates >0.5. Log-ratios of replicates were combined using the fusion algorithm. The resulting log-ratios were imported into Nexus-software Copy Number-V3.1 (BioDiscovery, US) to visualise and analyse copy number changes within or between subsets of SCC.
The confidence flag filter selection: A and B (= confidence > 0.7)
Postprocessing protocol:
- Normalization then exclusion
- Block median normalization excluding controls
Next, exclude spots if:
- ID = controls, THEN
- Confidence < 0.3, THEN
- Quality < 1, THEN
- Replicates with StdDEV > 0.5
Next:
- Replicates are combined based on “Name”, using “Fusion” algorithm
Remark: Although this software package includes algorithms that deal with the (local) background, the background values are not listed in any of the output files.
