|
| Status |
Public on Apr 17, 2018 |
| Title |
ctrl 24 hrs, 3 |
| Sample type |
RNA |
| |
|
| Source name |
HPCVE Uninfected 24 hrs in RPMI media Placenta 3
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Placenta
gestational age: Term
|
| Treatment protocol |
0,5 cm3 explants were incubated in RPMI Media with 105 o 106 T. cruzi trypomastigotes (Y strain) for 2 or 24 hrs. Explants were collected in RNALater (Thermo Fisher Scientific) and stored at -80°C until RNA isolation.
|
| Growth protocol |
HPCVE were incubated in RPMI media suplemented with 5% FBS and antibiotics (penicillin-streptomycin) at 37°C in a humid atmosphere at 5% CO2. Parasites (Trypomastigotes) from T. cruzi (Y Strain) were obtained from previously infected Vero cells (ATCC® CCL-81). The infective trypomastigotes were separated from cellular debris by low speed centrifugation (500 x g). From the supernatant, the parasites were isolated by centrifugation at 3500 x g, suspended in RPMI media and quantified in a Neubauer Chamber.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with the PureLink RNA Mini Kit (Thermo Fisher Scientific). RNA integrity was analyzed with a Bioanalyzer 2100 (Agilent Technologie) obtaining RNA Integrity numbers (RIN) above 8 for all samples. Quantification of total RNA was performed with Qubit RNA quantification kit(Themo Fisher Scientific).
|
| Label |
Cy3
|
| Label protocol |
100ng of total RNA were reverse-transcribed into cDNA, the transcribed to cRNA and Cy3-labeled with Low Input Quick Amp- One color Labeling Kit (Agilent Technologies). The labeled cRNA was purified with Illustra RNAspin Mini Isolation kit (GE Healthcare) and the total yield was measured with Qubit RNA HS kit (ThermoFisher Scientific)
|
| |
|
| Hybridization protocol |
Labeled samples were hybridized with SurePrint G3 Human GE 8x60K chips for 17 h at 60° C in an Agilent G2545A hybridization oven at 10 rpm with the instrucvions of Gene ExpressionHybridization Kit (Agilent).
|
| Scan protocol |
lides were washed with Gene Expression Wash Buffer 1 and 2 according to mannufacturer's instuctions and scanned immediately with an Agilent microarray scanner G2565BA. The parameters used were: Scan region Agilent HD (61 × 21.6 mm),Scan resolution 5 µm, Tiff file dynamic range 20 bit, Green PMT gain 100%.
|
| Description |
Gene expression after 24 hrs in RPMI media (uninfected control)
|
| Data processing |
Agilent Feature Extraction (version 9.5.1), was used for quality control, data filtering, and data normalization. Standar parameters and Grid Template 039494_D_F_20140813 were used. Extracted data from the SurePrintG3 8x60K chips were analyzed using GeneSpring GX 13.0 software
|
| |
|
| Submission date |
Apr 16, 2018 |
| Last update date |
Apr 17, 2018 |
| Contact name |
Ulrike Kemmerling |
| E-mail(s) |
ukemmerling@uchile.cl
|
| Phone |
+5629786261
|
| Organization name |
Universidad de Chile
|
| Department |
Faculty of Medicine
|
| Lab |
Anatomy and developmental biology
|
| Street address |
Independencia 1027
|
| City |
Santiago |
| ZIP/Postal code |
8380453 |
| Country |
Chile |
| |
|
| Platform ID |
GPL16699 |
| Series (1) |
| GSE113155 |
Transcriptome analysis by microarray of human placental chorionic villi explants infected with Trypanosoma cruzi |
|
