|
| Status |
Public on Dec 07, 2011 |
| Title |
P7.5C control |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
P7.5C
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: mouse kidney blastemal cells
age: 1 week old
|
| Extracted molecule |
total RNA |
| Extraction protocol |
kit Pico Pure RNA Isolation Kit
|
| Label |
Cy3
|
| Label protocol |
Labeled cDNA was generated in a reverse transcriptase reaction using 7mg of amplified RNA (aRNA), random hexamer primer (Invitrogen), Cy3- or Cy5-labeled dCTP (Amersham, Biosciences) and SuperScript II (Invitrogen).For reference, 4mg of aRNA were Cy3- or Cy5-labeled dCTP (Amersham, Biosciences) in the presence of SuperScript II (Invitrogen).
|
| |
|
| Channel 2 |
| Source name |
Reference
|
| Organism |
Homo sapiens |
| Characteristics |
reference: pool of 15 human cell lines (Daudi (Burkitt's lymphoma)
cell line: DLD-1 (colorectal adenocarcinoma)
cell line: DU 145 (prostate carcinoma)
cell line: FaDu (pharynx squamous cell carcinoma)
cell line: GM-637 (fibroblast)
cell line: H-146 (small cell lung carcinoma)
cell line: HT-1080 (fibrosarcoma)
cell line: HB4a (normal mammary luminal epithelium)
cell line: HEK293 (human embryonic kidney)
cell line: Jurkat (acute T cell leukemia)
cell line: Saos-2 (osteosarcoma)
cell line: SK-BR-3 (breast adenocarcinoma)
cell line: SK-MEL-28 (melanoma)
cell line: T24 (bladder transitional cell carcinoma)
cell line: T98G (glioblastoma multiform))
|
| Biomaterial provider |
Coriell; http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=GM00637
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol
|
| Label |
Cy5
|
| Label protocol |
Labeled cDNA was generated in a reverse transcriptase reaction using 7mg of amplified RNA (aRNA), random hexamer primer (Invitrogen), Cy3- or Cy5-labeled dCTP (Amersham, Biosciences) and SuperScript II (Invitrogen).For reference, 4mg of aRNA were Cy3- or Cy5-labeled dCTP (Amersham, Biosciences) in the presence of SuperScript II (Invitrogen).
|
| |
|
| |
| Hybridization protocol |
Hibridization - Solution: 5 X SSC; 0,1% SDS; 4 ug poly A DNA; 4 ug Cot1 DNA; 5X Denhardts ; 10 ug Esperma de salmao, Formamida 25%. / Temperature:14 a 24 hs / Time:42 C / Touch Down:
Washing - Round 1 - Solution: SSC2X / Temperature:42ºC / Time:15' / Washing Number: 1
Washing - Round 2 - Solution: SSC0,1X-SDS0,1% / Temperature:42ºC / Time:15' / Washing Number: 2
Washing - Round 3 - Solution: SSC0,1X / Temperature:42ºC / Time:15' / Washing Number: 2
|
| Scan protocol |
Hardware: Perkinelmer / Software: ScanArray
|
| Description |
This research was approved by Ethic Committee of the Medical and Research Center - Hospital A. C. Camargo number 764/06. Twenty-four sporadic and favorable histology advanced (Stage III or IV) Wilms tumor (WT) samples from Children Oncology Group (NWTSG), four human differentiated kidneys , and four temporal stages of kidney differentiation in mouse (E15.5, E17.5, P1.5 and P7.5) were used for this study. Metanephric blastema cells from four temporal differentiation stages, from E15.5 to the fully differentiated kidney in mouse, and blastemal cells of WT and differentiated kidneys in human were used for RNA extraction and amplification. RNA were extracted with PicoPure™ RNA Isolation kit (Arcturus Engineering #KT0204) and treated with RNase-Free DNase Set (Qiagen #79254; Qiagen-Germantown MD USA). Amplification methodology used RiboAmpTM RNA amplification KIT0201 and RiboAmpTM HS Plus KIT0505 (ARCTURUS) for human and mouse samples, respectively. A pool of RNAs obtained from 15 distinct human cell lines was amplified in the presence of SuperScript III and oligo dT(24)-T7. Amplified RNA was then used in a transcriptase reverse reaction into cDNA in the presence of Cy3 or Cy5-labeled dCTP. Dye-swap was performed for each sample as control for dye bias and used as replicate.
|
| Data processing |
R version 2.9.2 (2009-08-24), Lowess-normalized signal intensity, log2 test/reference
|
| |
|
| Submission date |
Dec 07, 2010 |
| Last update date |
Dec 13, 2011 |
| Contact name |
Renato David Puga |
| Organization name |
Universidade de São Paulo
|
| Department |
Instituto de Psiquiatria
|
| Lab |
Laboratório de Genética
|
| Street address |
Dr Ovidio Pires de Campos,785
|
| City |
São Paulo |
| State/province |
São Paulo |
| ZIP/Postal code |
05403-010 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL9692 |
| Series (1) |
| GSE25965 |
Transduction signaling signature of Wilms tumor revealed by parallel analysis of kidney differentiation |
|
