tissue: breast carcinoma sample type: formalin-fixed paraffin-embedded tissue
Treatment protocol
Excess paraffin was removed with a scalpel from either side of 40-60um scrolls. Sectioning samples reduces accumulation of debris during the sorting process. Each sectioned piece was collected into individual microcentrifuge tubes, then washed three times with 1 ml Xylene for 5 minutes to remove remaining paraffin. Each sample was rehydrated in sequential ethanol washes (100% 5 minutes x2, then 95%, 70%, 50% and 30% ethanol) and washed 2 times in 1ml 1 mM EDTA pH 8.0. A 1 ml aliquot of 1 mM EDTA pH 8.0 was added to the samples and incubated at 95oC for 80 minutes to facilitate the removal of protein cross-links present in FFPE tissue. Samples were then cooled to room temperature for > 5 minutes, followed by addition of 300 ul PBS pH 7.4 and gentle centrifugation for 2 minutes at 3.6 x g. The supernatant was carefully removed and the pellet washed three times with 1 ml PBS pH 7.4/0.5mM CaCl2 to remove EDTA. Each sample was digested overnight (6-17 hours) in 1ml of a freshly prepared enzymatic cocktail containing 50 units/ml of collagenase type 3, 80 units/ml of purified collagenase, and 100 units/ml of hyaluronidase in PBS pH 7.4/0.5mM CaCl2 buffer. Each enzyme was rehydrated with PBS pH 7.4/0.5mM CaCl2 buffer, then stored at -200C immediately prior to addition to the cocktail mixture. Following overnight digestion, 500 ul NST was added to each sample to facilitate pelleting. Samples were centrifuged for 5 minutes at 3000 x g, after which pellets were resuspended in 750 ul of NST/10% fetal bovine serum and then passed through a 25 G needle 10-20 times. The samples were filtered through a 35 um mesh and collected into a 5 ml Polypropylene round bottom tube. The mesh was rinsed with an additional 750 ul of NST/10% fetal bovine serum and placed on ice while processing remaining samples. The total volume in the tube for each sample was approximately 1.5 ml. An equal volume of 20ug/ml DAPI was added to each tube to achieve a final concentration of 10ug/ml DAPI prior to flow sorting with a BD Influx cytometer with ultraviolet excitation (Becton-Dickinson, San Jose, CA). The optimal settings for sorting FFPE samples with the Influx sorter were as follows: Drop formation was achieved with piezzo amplitude of 6-10 volts and a drop frequency of 30 khertz. The sort mode was set to purity yield with a drop delay of 31.5-32. Sheath fluid pressure was typically 17-18 psi with a 100 um nozzle. For single parameter DNA content assays, DAPI emission was collected at >450nm. DNA content and cell cycle were then analyzed using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA).
Growth protocol
None.
Extracted molecule
genomic DNA
Extraction protocol
DNA from sorted nuclei was extracted using an amended protocol from the QIAamp® DNA Micro Kit from Qiagen (Valencia, CA). Briefly, each sorted sample was resuspended in 180ul buffer ATL and 20ul proteinase K, then incubated for 3 hours at 56°C for complete lysis. Samples were bound and washed according to QIAamp® DNA Micro Kit instructions, eluted into 50ul of H20, then precipitated overnight with 5ul 3 M sodium acetate and 180 ul 100% EtOH. Each sample was then centrifuged for 30 minutes at 20,000 x g, and washed in 1 ml of 70% EtOH for 30 minutes at 20,000 x g. The samples were carefully decanted and the DNA pellet was dried by speed vacuum then resuspended in a small volume (e.g. 10-50ul) of H20 for final concentrations suitable for accurate quantification.
Label
Cy5
Label protocol
Genomic DNAs from sorted FFPE samples were amplified using the Ovation® WGA FFPE System from NuGEN® Technologies (San Carlos, CA). DNA was processed in accordance with the Ovation® WGA FFPE standard protocol with an alternate T7 endonuclease fragmentation step. Resulting amplified product was either used as template for aCGH analysis or processed with the Nugen Encore ds-DNA module according to the supplier's instructions in order to generate double-stranded end repaired DNA as input for library suitable for next-generation sequencing. Extracted fresh frozen sourced genomic DNA was amplified using the phi29 based Illustra GenomiPhi V2 Amplification kit from GE Healthcare Bio-sciences Corp (Piscataway, NJ) according to our published protocols. A 100 ng aliquot of pooled 46,XX DNA (Promega, Madison, WI) was amplified with the matching amplification protocol to generate a suitable reference for each aCGH experiment using amplified DNA template. In all cases, the quality of the amplification product was assessed by gel electrophoresis. Fresh frozen phi29 amplified and FFPE non-amplified DNAs were treated with DNAse 1 prior to Klenow-based labeling. High molecular weight phi29 templates were digested for 30 minutes while the smaller fragmented FFPE samples were digested for only 1 minute. In each case, 1ul of 10x DNase 1 reaction buffer and 2ul of DNase 1 dilution buffer were added to 7ul of DNA sample and incubated at room temperature, then transferred to 70°C for 30 minutes to deactivate DNase 1. In contrast, the amplified FFPE sourced DNAs do not require DNase 1 treatment prior to Klenow-based labeling. Sample and reference templates were then labeled with Cy-5 dUTP and Cy-3 dUTP, respectively, using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols.
Excess paraffin was removed with a scalpel from either side of 40-60um scrolls. Sectioning samples reduces accumulation of debris during the sorting process. Each sectioned piece was collected into individual microcentrifuge tubes, then washed three times with 1 ml Xylene for 5 minutes to remove remaining paraffin. Each sample was rehydrated in sequential ethanol washes (100% 5 minutes x2, then 95%, 70%, 50% and 30% ethanol) and washed 2 times in 1ml 1 mM EDTA pH 8.0. A 1 ml aliquot of 1 mM EDTA pH 8.0 was added to the samples and incubated at 95oC for 80 minutes to facilitate the removal of protein cross-links present in FFPE tissue. Samples were then cooled to room temperature for > 5 minutes, followed by addition of 300 ul PBS pH 7.4 and gentle centrifugation for 2 minutes at 3.6 x g. The supernatant was carefully removed and the pellet washed three times with 1 ml PBS pH 7.4/0.5mM CaCl2 to remove EDTA. Each sample was digested overnight (6-17 hours) in 1ml of a freshly prepared enzymatic cocktail containing 50 units/ml of collagenase type 3, 80 units/ml of purified collagenase, and 100 units/ml of hyaluronidase in PBS pH 7.4/0.5mM CaCl2 buffer. Each enzyme was rehydrated with PBS pH 7.4/0.5mM CaCl2 buffer, then stored at -200C immediately prior to addition to the cocktail mixture. Following overnight digestion, 500 ul NST was added to each sample to facilitate pelleting. Samples were centrifuged for 5 minutes at 3000 x g, after which pellets were resuspended in 750 ul of NST/10% fetal bovine serum and then passed through a 25 G needle 10-20 times. The samples were filtered through a 35 um mesh and collected into a 5 ml Polypropylene round bottom tube. The mesh was rinsed with an additional 750 ul of NST/10% fetal bovine serum and placed on ice while processing remaining samples. The total volume in the tube for each sample was approximately 1.5 ml. An equal volume of 20ug/ml DAPI was added to each tube to achieve a final concentration of 10ug/ml DAPI prior to flow sorting with a BD Influx cytometer with ultraviolet excitation (Becton-Dickinson, San Jose, CA). The optimal settings for sorting FFPE samples with the Influx sorter were as follows: Drop formation was achieved with piezzo amplitude of 6-10 volts and a drop frequency of 30 khertz. The sort mode was set to purity yield with a drop delay of 31.5-32. Sheath fluid pressure was typically 17-18 psi with a 100 um nozzle. For single parameter DNA content assays, DAPI emission was collected at >450nm. DNA content and cell cycle were then analyzed using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA).
Growth protocol
None.
Extracted molecule
genomic DNA
Extraction protocol
DNA from sorted nuclei was extracted using an amended protocol from the QIAamp® DNA Micro Kit from Qiagen (Valencia, CA). Briefly, each sorted sample was resuspended in 180ul buffer ATL and 20ul proteinase K, then incubated for 3 hours at 56°C for complete lysis. Samples were bound and washed according to QIAamp® DNA Micro Kit instructions, eluted into 50ul of H20, then precipitated overnight with 5ul 3 M sodium acetate and 180 ul 100% EtOH. Each sample was then centrifuged for 30 minutes at 20,000 x g, and washed in 1 ml of 70% EtOH for 30 minutes at 20,000 x g. The samples were carefully decanted and the DNA pellet was dried by speed vacuum then resuspended in a small volume (e.g. 10-50ul) of H20 for final concentrations suitable for accurate quantification.
Label
Cy3
Label protocol
Genomic DNAs from sorted FFPE samples were amplified using the Ovation® WGA FFPE System from NuGEN® Technologies (San Carlos, CA). DNA was processed in accordance with the Ovation® WGA FFPE standard protocol with an alternate T7 endonuclease fragmentation step. Resulting amplified product was either used as template for aCGH analysis or processed with the Nugen Encore ds-DNA module according to the supplier's instructions in order to generate double-stranded end repaired DNA as input for library suitable for next-generation sequencing. Extracted fresh frozen sourced genomic DNA was amplified using the phi29 based Illustra GenomiPhi V2 Amplification kit from GE Healthcare Bio-sciences Corp (Piscataway, NJ) according to our published protocols. A 100 ng aliquot of pooled 46,XX DNA (Promega, Madison, WI) was amplified with the matching amplification protocol to generate a suitable reference for each aCGH experiment using amplified DNA template. In all cases, the quality of the amplification product was assessed by gel electrophoresis. Fresh frozen phi29 amplified and FFPE non-amplified DNAs were treated with DNAse 1 prior to Klenow-based labeling. High molecular weight phi29 templates were digested for 30 minutes while the smaller fragmented FFPE samples were digested for only 1 minute. In each case, 1ul of 10x DNase 1 reaction buffer and 2ul of DNase 1 dilution buffer were added to 7ul of DNA sample and incubated at room temperature, then transferred to 70°C for 30 minutes to deactivate DNase 1. In contrast, the amplified FFPE sourced DNAs do not require DNase 1 treatment prior to Klenow-based labeling. Sample and reference templates were then labeled with Cy-5 dUTP and Cy-3 dUTP, respectively, using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols.
Hybridization protocol
All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE) prior to mixing and hybridization to 400k CGH arrays (Agilent Technologies, Santa Clara, CA) for 40 hours in a rotating 65°C oven.
Scan protocol
All microarray slides were scanned using an Agilent 2565C DNA scanner, and the images were analyzed with Agilent Feature Extraction version 10.7 using default settings.
Data processing
The aCGH data was assessed with a series of QC metrics, dye normalized and centralized, and then analyzed using an aberration detection algorithm (ADM2) which is included in Agilent Genomic Workbench 6.5 software. The latter identifies all aberrant intervals in a given sample with consistently high or low log ratios based on the statistical score derived from the average normalized log ratios of all probes in the genomic interval multiplied by the square root of the number of these probes. This score represents the deviation of the average of the normalized log ratios from its expected value of zero and is proportional to the height h (absolute average log ratio) of the genomic interval and to the square root of the number of probes in the interval.
