Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

Correction: Human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes

Human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes

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Comparison of normal neuroblasts with malignant neuroblastomas (low- and high-stage)

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disease state

Transcription profiling of human normal neuroblasts vs. malignant neuroblastomas (low- and high-stage)

Comparison of normal neuroblasts with malignant neuroblastomas (low- and high-stage)


Affymetrix:Protocol:Hybridization-[]

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-21526

P-MEXP-21559

1) mix cells with Lysing Matrix D microbeads (Qbiogene) and 700 µl RTL buffer (Qiagen) 2) homogenize using FastPrep FP220 (Qbiogene) 3) totalRNA isolation by Rneasy mini Qiagen kit (Qiagen)

P-MEXP-21560

Quality and integrity of the total RNA was controlled by running all samples on an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies; Waldbronn, Germany). For target synthesis starting from < 500 ng of total RNA (105 cells), an initial round of amplification was performed before synthesis of biotin-labeled aRNA. For the first round of amplification, synthesis of first- and second-strand cDNA was performed using the standard protocol provided by the manufacturer (Affymetrix). However, instead of proceeding to use the doublestranded cDNA in the biotin-labeled in vitro transcription reaction, the cDNA was resuspended in 8 µl of Rnase free water and used as a template to transcribe unlabeled antisense RNA (aRNA) using T7 RNA polymerase and the Promega P1300 RiboMax Kit (Promega). The reaction was incubated for 4 hours at 37°C and the resulting aRNA was purified using RNeasy spin columns (Qiagen). Eluted aRNA was precipitated by adding 0.1 volume of 7.5 mol/L ammonium acetate, 2.5 µg linear acrylamide (Ambion), and 2.5 volumes of 100% ethanol, and resuspended in 10 µl of RNase-free water. A second round of amplification was initiated by using the aRNA as template. After annealing aRNA with 0.7 µmol/L random hexamers (Pharmacia) for 10 minutes at 70°C, the mixture was chilled on ice and extended in a 20-µl reaction containing 4 µl of 5x first-strand reaction buffer, 2 µl of 0.1 mol/L dithiothreitol, 1 µl of 10 mmol/L dNTPs, and 1 µl of Superscript II. After 1-hour incubation at 42°C, 1 µl of 2 U/ml of RNase H was added, incubated for 20 minutes at 37°C, and inactivated at 95°C for 5 minutes. The resulting first-strand cDNA was annealed to 100 pmol of high performance liquid chromotography (HPLC)-purified T7T24 primer (EUROGENTEC) for 10 minutes at 70°C. Then, second-strand cDNA synthesis was performed by adding 90 µl of RNase-free water, 30 µl of 5x second-strand reaction buffer, 3 µl of 10 mmol/L dNTPs, 10 U DNA ligase and 40 U DNA polymerase. After incubating the second-strand cDNA reaction for 2 hours at 16°C, 20 U of T4 DNA polymerase were added, followed by incubation at 16°C for 10 minutes.The second-strand cDNA synthesis was stopped by adding 10 µl of 0.5 mol/L ethylenediaminetetraacetic acid. Double-stranded cDNA was purified by phenol: chloroform:isoamyl alcohol extraction using phase-lockgel (Eppendorf), precipitated with 0.5 volumes of 7.5 mol/L ammonium acetate and 2.5 volumes of 100% ethanol, and resuspended in 12 µl of RNase-free water. The concentration of biotin-labeled aRNA was determined by UV absorbance. In all cases, 10 µg of each biotinylated aRNA preparation were fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer.  

P-MEXP-22407

normalisation of raw CEL files using RMA (robust multi chip average method) in bioconductor (R)

P-MEXP-22577

Reagents  1. 0.5 M NaOH (VWR International, VELC8697) 2. Glass slides: uncoated or poly-L-lysine (e.g. Superfrost plus microscope slides  from BDH, 406/0179/00) 3. Aluminium foil 4. RNase free glass recipients for diluting solutions and staining slides 5. Tissue-Tek OCT Compound (Sakura, 4583) 6. Nuclease-free H2O (Sigma, W4502) 7. EtOH absolute (VWR International, VELC1115) 8. EosinY (Aldrich, 11,983-0) 9. Glacial acetic acid (VWR International, VELC1005) 10. Eosin Y solution = 0.4g eosin Y in 40 ml nuclease-free H2O, add 120 ml 95% EtOH, add 0.8 ml glacial acetic acid 11. Mayer’s hematoxylin solution (0.1%) (Sigma Diagnostics, MHS-16) 12. Xylene (VWR International, VELC1864) 13. RNeasy Mini Kit (50) (Qiagen, 74104) 14. RNase-free DNase Set (50) (Qiagen, 79254) 15. RiboGreen RNA quantification kit (Molecular Probes, R-11490) 16. RNA 6000 Pico LabChip Kit (Agilent, 5065-4476)  Method  Preparation of solutions and recipients RNASE-FREE: * Always work with gloves! * Overnight (=night before) treatment of cryotome knife in 0.5 M NaOH, wash with nuclease-free H2O before use. * Night before sectioning and staining: Bake the glass recipients and glass slides at 180°C for at least 6 hours (wrap glass slides and recipients in aluminium foil to avoid contact after baking). * Solutions as EtOH, H2O and xylene should be used with care. If possible, use a newly opened bottle of the solutions. Make dilutions of these solutions in RNase-free recipients with nuclease-free H2O.  Cryo-sections: * Snap-frozen tissue (liquid nitrogen) (can be stored at -80°C) * Embed in OCT on the cryotome tissue holder (don’t let defrost the tissue) * Make 7 µm cryo-sections (with knife that is pre-treated overnight in 0.5 M NaOH and washed with nuclease-free H2O) * Put on RNase-free glass slide (uncoated or poly-L-lysine coated)  Heamatoxylin-eosin staining: * Immediately after making the section: put slide in 70% EtOH (at least 30’’, up to 5’). * Nuclease-free H2O: rinse for 5’’ * Mayer’s heamatoxylin 30’’  * Nuclease-free H2O: rinse for 5’’ * 70% EtOH: 60’’ * 95% EtOH: 60’’ * Eosin Y solution: 30’’  * 95% EtOH: 60’’ * 100% EtOH: 60’’ * 100% EtOH: 60’’ (second jar) * xylene: 3’ * xylene: 5’ (second jar!) * Shake off excess and drain slide carefully by touching the corner to a particle free paper towel or tissue * Air dry at least 2’ * Store dry and dust free and try to go ahead with laser capture microdissection as soon as possible  LCM and RNA isolation: * Perform LCM under RNase-free conditions * Place cap in 500 µl microcentrifuge tube with RNA extraction buffer (see protocol RNeasy mini kit, Qiagen), turn upside down. You can store from this stage in freezer if you can not proceed immediately with the RNA isolation protocol. * Vortex for 2 minutes * Perform RNA isolation with RNeasy mini kit according to manufacturer protocol and include DNase step * Measure RNA concentration using the PicoGreen reagent on a fluorometer * Determine RNA quality with the 2100 Bioanalyzer (Agilent) using the RNA 6000 Pico LabChip Kit  

Transcription profiling of human normal neuroblasts vs. malignant neuroblastomas (low- and high-stage)

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