|
| Status |
Public on Sep 15, 2007 |
| Title |
HDC-A_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
B16-F10 HDC-A experimental tumors, pooled samples Nr.1-2
|
| Organism |
Mus musculus |
| Characteristics |
The B16-F10 mouse melanoma cell line was stable transfected to express an antisense RNA targeting the mouse L-histidine decarboxylase (HDC) mRNA. This transgenic B16-F10 subclone, designated as B16-F10 HDC-A, displays diminished HDC expression and histamine secretion.
Experimental mouse melanoma tumors were induced by grafting 2x10E+5 B16-F10 HDC-A cells to 8-12 weeks old C57BL/6 female mice. At 15 days after grafting, mice were sacrificed, and tumors were excised.
This channel sample consists of a pooled total RNA of two experimental tumors.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation was carried out from using RNeasy columns (Quiagen). RNA yield and purity were determined by an ND1000 spectrophotometer (Nanodrop). RNA integrity was checked by capillary electrophoresis using an RNA Series II 6000 Nano Kit (Agilent) and a 2100 Bioanalyzer (Agilent).
|
| Label |
Cy5
|
| Label protocol |
One ug of tumor-derived, pooled total RNA was spiked by an RNA Spike-In Kit (Agilent), reverse transcribed by a Low RNA Input Linear Amplification Kit (Agilent), and used for Cyanine 5-CTP-labeled (Perkin Elmer) cRNA synthesis in a linear amplification reaction. Successful labeling, cRNA yield and purity were controlled on an ND1000 (Nanodrop) spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
B16-F10 HDC-M cell line cultured in vitro, uniform reference sample
|
| Organism |
Mus musculus |
| Characteristics |
The mock-transfected subclone of the original B16-F10 mouse melanoma cell line is designated as B16-F10 HDC-M.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation was carried out from 1x10E+6 B16-F10 HDC-M cells using RNeasy columns (Quiagen). RNA yield and purity were determined by an ND1000 spectrophotometer (Nanodrop). RNA integrity was checked by capillary electrophoresis using an RNA Series II 6000 Nano Kit (Agilent) and a 2100 Bioanalyzer (Agilent).
|
| Label |
Cy3
|
| Label protocol |
One ug of cell line-derived, pooled total RNA was spiked by an RNA Spike-In Kit (Agilent), reverse transcribed by a Low RNA Input Linear Amplification Kit (Agilent), and used for Cyanine 3-CTP-labeled (Perkin Elmer) cRNA synthesis in a linear amplification reaction. Successful labeling, cRNA yield and purity were controlled on an ND1000 (Nanodrop) spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
750 ng Cyanine 5-CTP-labeled cRNA per pooled tumor sample was mixed with an equal amount of Cyanine 3-CTP–labeled uniform reference cRNA, and hybridized to 44K Whole Mouse Genome Oligo Microarrays (Agilent) according to the manufacturer's protocol: http://www.chem.agilent.com/scripts/LiteraturePDF.asp?iWHID=46782
|
| Scan protocol |
Array scanning, feature extraction and data normalization were done by Agilent’s DNA Microarray Scanner and Feature Extraction Software 8.5 (Agilent).
|
| Description |
This array slide was evaluated as part of an experiment based on a two-color experimental design, comparing individual pooled tumor samples via the uniform reference sample.
|
| Data processing |
Scanned data was transferred for statistical evaluation in the GeneSpring software package (Agilent) with default normalization scenario for Agilent two-color arrays. Identification of gene sets differentially expressed between HDC-A-, HDC-M- and HDC-S-transfected tumor groups was carried out by One-Way ANOVA with Benjamini-Hochberg multiple testing correction. Tukey’s All Pairwise Multiple Comparison was applied as post hoc test
|
| |
|
| Submission date |
Jul 20, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Zoltan Pos |
| E-mail(s) |
pos.zoltan@outlook.com.com
|
| Phone |
+(36)12102930-56435
|
| Organization name |
Semmelweis University
|
| Department |
Dept. of Genetics, Cell- and Immunobiology
|
| Street address |
4 Nagyvarad ter
|
| City |
Budapest |
| ZIP/Postal code |
H-1089 |
| Country |
Hungary |
| |
|
| Platform ID |
GPL2872 |
| Series (1) |
| GSE8541 |
Evaluation of the impact of neoplastic histamine secretion on melanoma progression |
|
