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Status |
Public on Mar 29, 2014 |
Title |
notochord_2 (mRNA) |
Sample type |
RNA |
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Source name |
notochord,sample 2
|
Organism |
Homo sapiens |
Characteristics |
gender: Male age: 25 weeks tissue: embryo intervertebral disc
|
Treatment protocol |
Paraffinsections from the fixed chordoma tissues were cut at 5 mm andstained with hematoxylin and eosin (H&E). All tissues were separated using laser capture microdissection(LCM)
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Growth protocol |
All thesamples were stored at -80℃ until they were processed for RNA extraction in 1 batch
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated from PFPE samples by usingRecoverAllTM Total Nucleic Acid Isolation Kit (Life Technologies, Carlsbad, CA, USA) for mRNA and miRNA microarrayanalysis, according to the manufacturer’s instructions.Sample purity and integrity was confirmed by A260/A280 spectrophotometry and capillary gel electrophoresis on a Bioanalyzer 2100 (Agilent)
|
Label |
biotin
|
Label protocol |
Total RNA per sample was amplified using a SensationPlus™ FFPE Amplification and WT Labeling Kit (Affymetrix). Samples were subjected to the first strand cDNA synthesis, promoter synthesis, subsequent in vitro transcription of senseRNA, double-stranded cDNA synthesis, and labeling of cDNA. Total RNA was reverse transcribed to form the cDNA, purified with Purification Beads subsequently. Total cDNA was poly (dA) tailed on the 3´ end. A T7 dT oligo with a 3´ blocking group is hybridized to the 3´ (dA) tail. klenow and dNTP mix generate a double stranded T7 promoter region on the single stranded cDNA. The promoter-modified cDNA was in vitro transcribed to form RNA. The senseRNA was purified with purification beads and reverse transcribed using random primer mix. second strand cDNA was made with klenow and RNase H. Finally, the double-stranded cDNA was purified, fragmented and end-labeled
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Hybridization protocol |
Samples were prepared for hybridization using the GeneChip Hyb Wash and Stain Kit (Affymetrix); mixed with Control Oligonucleotide B2, Eukaryotic Hybridization Controls and Hybridization Mix from and loaded to Human Gene ST 1.0 Arrays (Affymetrix). Hybridization was done in a GeneChip Hybridization Oven 640 (Affymetrix) for 17 hours, at +45°C, 60 rpm. Arrays were washed and PE-labeled on a GeneChip Fluidics Station 450 (Affymetrix)
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Scan protocol |
Arrays were scanned on a GeneChip Scanner 3000 7G with autoloader (Affymetrix). Raw data were generated with Affymetrix GeneChip Command Console
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Description |
gene expression data from notochord IVD of sample 2
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Data processing |
Raw data from the generated .CEL files were imported into Affymetrix Expression Console using the RMA method
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Submission date |
Mar 25, 2014 |
Last update date |
Mar 29, 2014 |
Contact name |
zhongjun Liu |
E-mail(s) |
starsky298@gmail.com
|
Phone |
15281085496
|
Organization name |
Peking University Third Hospital
|
Department |
Department of Orthopedics
|
Lab |
Spine tumor center
|
Street address |
49 North Garden Rd, Haidian, 100191, Beijing, China.
|
City |
Beijing, China |
State/province |
Beijing |
ZIP/Postal code |
100191 |
Country |
China |
|
|
Platform ID |
GPL10739 |
Series (1) |
GSE56183 |
Integrated miRNA-mRNA analysis revealing the potential roles of miRNAs in chordomas |
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