10 protocols
AccessionType
labeling
500 hundred nanograms of total RNA from each sample (either rim or core) and human universal reference RNA (Stratagene, La Jolla, CA) underwent one round of linear amplification in RiboAmp RNA Amplification kit. Sample cDNA was labeled with Cyanine 5 (Cy5) while reference RNA was labeled with Cyanine 3(Cy3) - CTP (PerkinElmer, Boston, MA) and cDNA concentration was assessed spectro-photometrically (GeneQuant II, Bottom Ltd., Cambridge, UK). Any remaining unamplified sample RNA was saved for validation studies.
nucleic_acid_extraction
Tumors are embedded in OCT, then sectioned in a cryostat at -20C, to a thickness of 8-10 um and placed onto HistoGene slides. Sections to be microdissected are removed from the -80C fixed and stained with an abbreviated Hematoxylin and Eosin protocol. Two thousand tumor core and invasive cells are dissected onto separate caps using the PixCell II instrument using CapSure™ Macro LCM Caps. The CapSure™ Macro LCM Caps LCM 0211 should be used with the AutoPix instrument. Cells in the tumor core are identified by nuclear atypia and size and captured using the larger spot sizes. Tumor cells immediately adjacent to necrotic areas, cortical areas, cells with small regular nuclei, endothelial and blood cells should be avoided. Individual white matter invading GBM cells can be identified by means of their nuclear atypia and heteropyknotic staining, which is consistent with that of the cells within the tumor core. They should be microdissected using the 7.5 um laser spot size and the initial power settings recommended by the PixCell II manual. After microdissection all harvested material should immediately be lysed on the cap by applying the lysis buffer (XB) from the PicoPure RNA Isolation Kit according to the PicoPure protocol and stored -80C. Cell populations harvested on different caps can be pooled at the time of RNA isolation. RNA integrity is varified by identification of distinct 28S and 18S ribosomal bands with an Agilent Bioanalyzer using the RNA 6000 Nano LabChip kit. Total RNA was isolated from 2000 LCM cells (to ensure at least 10 ng total RNA) using the PicoPure RNA solation Kit, following manufacturers protocol. mRNA is reverse transcribed with the RiboAmp RNA Amplification kit.
bioassay_data_transformation
ID_REF = Agilent Probe ID
VALUE = log2 of PRE_VALUE; normalized, log2 (test/ref) ratio
PRE_VALUE = normalized (test/ref) ratio
bioassay_data_transformation
ID_REF = Aglient probe ID
VALUE = log2 of PRE_VALUE; normalized, log2 (test/ref) ratio
PRE_VALUE = normalized (test/ref) ratio
bioassay_data_transformation
ID_REF =
VALUE = log2 of PRE_VALUE; normalized, log2 (test/ref) ratio
PRE_VALUE = normalized (test/ref) ratio
feature_extraction
Following hybridization, slides are washed, scanned and quantitated with the Axon GenePix 4000 microarray reader (Axon Instruments). Gene expression results are analyzed using GeneSpring (Silicon Genetics) software. Intensity dependent normalization is applied, where the ratio is reduced to the residual Lowess fit of the intensity versus ratio curve. The measured intensity of each gene is divided by its reference channel (signal from the universal reference RNA) in each sample. When the reference channel is below 10, the data point is considered uninformative. The ratios (sample over reference) for the tumor core experiments and invasive rim experiments are averaged and compared. Genes that are more than two-fold differentially regulated in the majority of the matched core/invasive rim sets are selected.
nucleic_acid_extraction
sample ordered from Stratagene as purified reference RNA
hybridization
As recommended by Agilent
image_aquisition
Agilent DNA microarray scanner and Feature Extraction Software with default settings for oligonucleotide expression arrays were used to capture and process array images.
nucleic_acid_extraction
sample order from Stratagene as purified reference RNA