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Status |
Public on Nov 26, 2014 |
Title |
C-01-1944 |
Sample type |
RNA |
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Source name |
Bone Marrow
|
Organism |
Homo sapiens |
Characteristics |
diagnosis: AML tissue: bone marrow treatment status: Pre-treatment
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted using the TRIzol® Plus RNA Purification Kit (Life Technologies, Grand Island NY) following the manufacturer's instructions. mRNA was purified from the total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). RNA quantity and quality were measured by NanoDrop ND-1000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis.
|
Label |
Cy3
|
Label protocol |
Sample labeling was performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, each mRNA sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing random priming. The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000.
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Hybridization protocol |
1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer. The mixture was heated at 60 °C for 30 min. 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.
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Scan protocol |
The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C). Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images.
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Data processing |
Quantile normalization and subsequent data processing were performed with using Agilent GeneSpring GX v11.5 software package (Agilent Technologies). After quantile normalization of the raw data, LncRNAs and mRNAs that at least 2 out of 4 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis. Differentially expressed LncRNAs and mRNAs were identified through Fold Change and T-test filtering.
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Submission date |
Nov 25, 2014 |
Last update date |
Nov 26, 2014 |
Contact name |
Ramiro Garzon |
E-mail(s) |
Ramiro.Garzon@osumc.edu
|
Organization name |
Ohio State University
|
Street address |
460 12th street
|
City |
Columbus |
ZIP/Postal code |
43212 |
Country |
USA |
|
|
Platform ID |
GPL16956 |
Series (1) |
GSE63614 |
Expression and Prognostic impact of LncRNAs in AML |
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