|
| Status |
Public on Feb 28, 2010 |
| Title |
MIA_AMBX_Rep3 |
| Sample type |
RNA |
| |
|
| Source name |
MIA_AMBX
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Mia-PaCa-2
tissue type: pancreatic carcinoma
|
| Biomaterial provider |
American Type Culture Collection (ATCC, Manassas, VA)
|
| Treatment protocol |
Amblyomin-X was added to a final concentration of 1 uM and cells were incubated for 4 hours prior to RNA isolation.
|
| Growth protocol |
Cells were cultivated in RPMI 1640 medium (75cm2 flasks) containing 10% (v/v) FBS, and incubated at 37°C and 5% CO2 in humid atmosphere, under standard procedures.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from Amblyomin-X-treated and untreated control cells using the TRIzol reagent (Invitrogen, Carlsbad, CA) as recommended by the manufacturer. The integrity of RNA samples was assessed by analysis of 28S/18S rRNA ratios following capillary electrophoresis (2100 Bioanalyser, Agilent Technologies Inc), which should be equal or greater than 1.5.
|
| Label |
biotin-NTP
|
| Label protocol |
Target labeling and hybridization was performed strictly as recommended by the array manufacturer (GE Healthcare, UK). In brief, the purified and high-quality total RNA was used in reverse transcription reactions to convert messenger RNA (mRNA) in double-stranded cDNA prior to biotin labeling. cRNA was synthesized by in vitro transcription of cDNA and simultaneously labeled with biotin-NTP mix. The samples were filtered to recover biotinilated cRNA.
|
| |
|
| Hybridization protocol |
Labeled cRNA targets were fragmented at 94ºC for 20 minutes prior hybridization with arrays at 37ºC during 18 hours. After the hybridization, the bioarrays were washed and bound targets were detected following incubation with Cy5-Streptavidin (GE Healthcare, UK). The bioarrays were washed, dried and protected from light. All samples processed in parallel and the arrays were incubated simultaneously using the same working solution to limit technical variation across the experiments.
|
| Scan protocol |
Following the hybridization, bioarrays were scanned immediately in a an arrayWoRx scanner (Applied Precision Inc., WA, USA).
|
| Description |
MIA_AMBX_Rep3_NORM
|
| Data processing |
CodeLink Expression Analysis software (GE Healthcare) was used to extract background-subtracted spot intensities from microarray images.
To make experiments comparable, intensity data from different hybridizations were normalized by the quantile method.
|
| |
|
| Submission date |
Sep 15, 2009 |
| Last update date |
Sep 17, 2009 |
| Contact name |
Eduardo Moraes Reis |
| E-mail(s) |
emreis@iq.usp.br
|
| Phone |
+55-11-30912173
|
| Organization name |
University of São Paulo
|
| Department |
Biochemistry
|
| Street address |
Av. Prof. Lineu Prestes, 748
|
| City |
São Paulo |
| State/province |
SP |
| ZIP/Postal code |
05508-900 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL2895 |
| Series (1) |
| GSE18124 |
A new tick Kunitz type inhibitor, Amblyomin-X, induces tumor cell death by modulating genes related to the cell cycle and targeting the ubiquitin-proteasome system |
|
