|
| Status |
Public on Jun 29, 2016 |
| Title |
RCCPDX13 model at passage P4 |
| Sample type |
RNA |
| |
|
| Source name |
Tumor tissue of the RCCPDX13 model at P4
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: CCC human tumor
passages: P4
|
| Treatment protocol |
There were no prior treatment.
|
| Growth protocol |
Fresh tumor tissue were harvested at P0 (primary tumor) directly after surgery, and at the different passages in anesthetized mice (gazeous anesthesia)as indicated in the Samples section and prior to mice euthanasia.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from patients’ primary tumors and from corresponding tumors at passage ranging from P1 to P8 was obtained using Qiagen RNAeasy mini columns according to manufacturer’s protocol. The concentration and integrity/purity of each RNA sample were measured using RNA 6000 LabChip kit (Agilent) and the Agilent 2100 bioanalyzer.
|
| Label |
Biotin
|
| Label protocol |
The 3’IVT protocol underwent reverse transcription, primed with T7 oligo(dT) primer, of total RNA to synthesize first-strand cDNA containing a T7 promoter sequence. This cDNA was then converted into a double-stranded DNA template for transcription. The second-strand cDNA synthesis converted the single-stranded cDNA into a double stranded DNA template for transcription. The reaction employed DNA polymerase and RNase H to simultaneously degrade the RNA and synthesize second strand cDNA. In vitro transcription synthesized aRNA and incorporated a biotin-conjugated nucleotide. The aRNA was then purified to remove unincorporated NTPs, salts, enzymes, and inorganic phosphate. Fragmentation of the biotin‑labeled aRNA prepared the sample for hybridization onto Affymetrix GeneChip® arrays (U133 Plus 2.0). The quality of fragmented PCR products was checked on 1% agarose gel.
|
| |
|
| Hybridization protocol |
Arrays were washed, stained (fluidics: FS450 with FS450_0001 protocol).
|
| Scan protocol |
Scanned using a GeneArray scanner (GeneChip® Scanner 3000)
|
| Description |
Gene expression data from tumor tissue
|
| Data processing |
Scans were processed with Affymetrix GeneChip® Command Console (AGCC 3.2.3) (INS GEN/0036*). The scanned images were converted into numerical values of the signal intensity (Signal) and into categorical expression level measurement (Absolute Call) using the Affymetrix MAS 5.0 files. Image QCs were performed by visual inspection (INS GEN/0008). Array QC metrics were determined using the QC tool (R script created from historical data and Affymetrix recommendations) with the following thresholds: “Z”-parameter (Z-BG, Z-Scale, Z-Present, Z-Actin, Z-GAPDH, Z-Deg): A Z-score is calculated for each QC metrics as follows [Z = (x – mean) / standard deviation]. The Z-score provide a way to assess “outlier” behaviour (too good or too bad relative to the mean).
|
| |
|
| Submission date |
Jun 28, 2016 |
| Last update date |
Jun 29, 2016 |
| Contact name |
Thierry Massfelder |
| Organization name |
INSERM
|
| Street address |
11 rue Humann
|
| City |
Strasbourg |
| ZIP/Postal code |
67085 |
| Country |
France |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE83820 |
Study of the molecular stability of patient-derived tumor xenograft (PDX) for human renal cell carcinoma (RCC) in nude mice during subsequent passages in mice |
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