|
| Status |
Public on Apr 27, 2022 |
| Title |
Xenograft treated with Crenigacestat 1 |
| Sample type |
RNA |
| |
|
| Source name |
iCCA xenograft mice model
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Xenograft tumor
treatment: Crenigacestat (8 mg/kg)
host strain: CD1 immunodeficient nude mouse
|
| Growth protocol |
5*106 HUCCT1 cells were injected subcutaneously into the right flank of CD1 immunodeficient nude mouse. 2-3 weeks after injection, when tumor masses reached approximately reach the volume of 60-70 mm3, mice were allocated in two experimental groups: a group treated orally (gavage) with vehicle and the other one treated orally (gavage) with Crenigacestat
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation from tissue was performed using TRIzol® (Thermo Fisher Scientific,Waltham, MA, USA) in combination with TissueLyser homogenizer (Qiagen, Hilden, Germany) according to manufacturer’s instructions. In addition, a column-based purification step has been done by using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and total RNA was then eluted in ribonuclease-free water. RNA concentration and quality were determined with the NanoDrop Spectrophotometer (Thermo Fisher Scientific, MA, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), respectively.
|
| Label |
biotin
|
| Label protocol |
One hundred ng of total RNA total RNA were subjected to two cycles of cDNA synthesis with the Affymetrix WT PLUS expression Kit. The first cycle – first strand synthesis is performed using an engineered set of random primers that exclude rRNA-matching equences and include the T7 promoter sequences. After second-strand synthesis, the resulting cDNA is in vitro transcribed with the T7 RNA polymerase to generate a cRNA. This cRNA is subjected to a second cycle – first strand synthesis in the presence of dUTP in a fixed ratio relative to dTTP. Single strand cDNA is then purified and fragmented with a mixture of uracil DNA glycosylase and apurinic/apirimidinic endonuclease 1 (Affymetrix) in correspondence of incorporated dUTPs. DNA fragments are then terminally labeled by terminal deoxynucleotidyl transferase (Affymetrix) with biotin.
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| |
|
| Hybridization protocol |
The biotinylated DNA was hybridized to the GeneChip Human Transcriptome Array 2.0 Arrays (Affymetrix), containing containing more than 286263 full length transcripts covering 44,699 coding genes and 22,829 non coding genes selected from Homo sapiens genome databases RefSeq, ENSEMBL and GenBank.
|
| Scan protocol |
Chips were washed, stained and scanned on the Affymetrix Complete GeneChip® Instrument System (Affymetrix, Santa Clara, CA), generating digitized image data and raw intensity data.
|
| Data processing |
The raw data were preprocessed with background correction and normalization with the Affymetrix Expression Console. The Affymetrix Transcriptome Analysis Console 4.0 was used to identify significantly differential expressed genes using the Limma eBayes method.
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| |
|
| Submission date |
May 07, 2020 |
| Last update date |
Apr 27, 2022 |
| Contact name |
Grazia Serino |
| E-mail(s) |
[email protected]
|
| Organization name |
National Institute of Gastroenterology “S. de Bellis”, Research Hospital
|
| Street address |
Via Turi, 27
|
| City |
Castellana Grotte (BA) |
| ZIP/Postal code |
70013 |
| Country |
Italy |
| |
|
| Platform ID |
GPL17586 |
| Series (1) |
| GSE150024 |
Effect of Crenigacestat treatment in xenograft model of HUCCT1 intrahepatic cholangiocarcinoma |
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