|
| Status |
Public on Feb 27, 2007 |
| Title |
universal_reference_vs_6_cell_lines_(Daudi) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Human universal reference RNA
|
| Organism |
Homo sapiens |
| Characteristics |
Human Universal RNA (Clontech, USA) is a mixture of RNA from several tissues.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Human Universal RNA were amplified with RiboAmp RNA Amplification kit (ARCTURUS ,USA). Quality of polyA+RNA and contamination of ribosomal RNA were assessed using Agilent Lab-on-a- Chip 2100 Bioanalyser (Agilent Technologies, USA).
|
| Label |
Cy3
|
| Label protocol |
1microgram of polyA+universal reference RNA (ribosomal RNA contamination subtracted) and lamba-A RNA for spiked control were subjected to fluorescent labeling with Cy3-dUTP using Cyscribe First- Strand cDNA Labeling kit(Amersham).
|
| |
|
| Channel 2 |
| Source name |
human B cell line Daudi
|
| Organism |
Homo sapiens |
| Characteristics |
Cell line: Daudi, Tissue :Burkitt's lymphoma
|
| Growth protocol |
Human cell lines were maintained in RPMI1640 supplemented with 10% heat-inactivated FCS, sodium pyruvate, non- essential amino acids solution, L-gulutamine, and 2-mercaptoethanol.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
PolyA+RNA were isolated from cells using mTRAP mRNA isolation Midi kit(Active Motif, USA). Quality of polyA+RNA and contamination of ribosomal RNA were assessed using Agilent Lab-on-a- Chip 2100 Bioanalyser(Agilent Technologies, USA).
|
| Label |
Cy5
|
| Label protocol |
1ug of polyA+RNA from cells(ribosomal RNA contamination subtracted) and lamba-A RNA for spiked control were subjected to fluorescent labeling with Cy5-dUTP using Cyscribe First- Strand cDNA Labeling kit(Amersham).
|
| |
|
| |
| Hybridization protocol |
Labeled probes were mixed together and loaded onto the microarray and subjected to 65℃ incubation for 16-20hours. The microarray was subsequently washed in 2xSSC, 2xSSC/ 0.2%SDS at55℃ for 5min twice, 2xSSC/0.2%SDS at 65℃ for 5min, 0.05xSSC at room temperature ,and spun for 3min at 600rpm to dry.
|
| Scan protocol |
Fluorescence image of the microarray was obtained using Affymetrix 428 Array Scanner. Image intensity data were extracted with ImaGene software(BioDiscovery).
|
| Description |
Gene expression profile of glycan-related genes
|
| Data processing |
Spot intensities were corrected for local background and log2 value of Cy5/Cy3 were calculated. The normalized ratio was obtained by calculating the sigma of each channel. To judged spot reliability, signal intensities were corrected for local background and spot intensity exceeding threshold value {=Average(back)+2*STDEV(back)} was ruled reliable for each channel. Rank 5 stands for being reliable in both channel, rank 4 for single channel, rank 3 for below threshold in both, rank 2 for taken off from further calculation due to negative value of signal, rank 1 taken off from further calculation due to the noise on the scanned array image.
|
| |
|
| Submission date |
Mar 08, 2006 |
| Last update date |
Feb 27, 2007 |
| Contact name |
Hiromu Takematsu |
| E-mail(s) |
[email protected]
|
| Organization name |
Kyoto Univ. Sch. of Medicine
|
| Department |
Human Health Science
|
| Lab |
Biochemistry
|
| Street address |
Shogoin-Kawahara 53
|
| City |
Kyoto |
| State/province |
Kyoto |
| ZIP/Postal code |
606-8507 |
| Country |
Japan |
| |
|
| Platform ID |
GPL3465 |
| Series (1) |
| GSE4407 |
Comparison of the expression profiles of human B cell lines. |
|
