|
| Status |
Public on Feb 27, 2015 |
| Title |
ASH2L_KD_SH272_DHT_12HRS_1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
LNCaP, ASH2L shRNA and DHT
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP
cell type: prostate cancer cell line
treatment: ASH2L shRNA and DHT
|
| Treatment protocol |
Cells were plated in 100mM plates at a desired concentration and infected with shRNA or siRNA targeting ASH2L, Menin or MLL1 vs. a non-targeting shRNA or siRNA. Knockdown efficiency was determined by qPCR.
|
| Growth protocol |
VCaP and LNCaP cells were grown in DMEM-Glutmax (Invitrogen) and RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, respectively.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol and an RNeasy Kit (Invitrogen) with DNase I digestion according to the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
10 ug of total RNA were primed with 2 ul of 100 uM T16N2 DNA primer at 70_C for 10 min, then reversed transcribed at 42_C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 uM each dATP, dTTP, dGTP, with 25 uM dCTP, 25 uM Cy3-label.
|
| |
|
| Channel 2 |
| Source name |
LNCaP, GIPZ and ethanol
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP
cell type: prostate cancer cell line
treatment: GIPZ and ethanol
|
| Treatment protocol |
Cells were plated in 100mM plates at a desired concentration and infected with shRNA or siRNA targeting ASH2L, Menin or MLL1 vs. a non-targeting shRNA or siRNA. Knockdown efficiency was determined by qPCR.
|
| Growth protocol |
VCaP and LNCaP cells were grown in DMEM-Glutmax (Invitrogen) and RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, respectively.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol and an RNeasy Kit (Invitrogen) with DNase I digestion according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
10 ug of total RNA were primed with 2 ul of 100 uM T16N2 DNA primer at 70_C for 10 min, then reversed transcribed at 42_C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 uM each dATP, dTTP, dGTP, with 25 uM dCTP, 25 uM Cy3-label.
|
| |
|
| |
| Hybridization protocol |
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially.
|
| Scan protocol |
Scanned on an Agilent G2505B scanner. Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
|
| Data processing |
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and LOWESS normalization. Control Features were omitted. The LogRatio columns from each feature file were used for further analyses.
|
| |
|
| Submission date |
Aug 27, 2014 |
| Last update date |
Feb 27, 2015 |
| Contact name |
Marcin Piotr Cieslik |
| E-mail(s) |
marcin.cieslik@gmail.com
|
| Organization name |
University of Michigan
|
| Department |
Pathology
|
| Lab |
MCTP
|
| Street address |
500 S State St
|
| City |
Ann Arbor |
| State/province |
Michigan |
| ZIP/Postal code |
48104 |
| Country |
USA |
| |
|
| Platform ID |
GPL6480 |
| Series (2) |
| GSE60836 |
Targeting the MLL complex in castration-resistant prostate cancer [expression] |
| GSE60842 |
Targeting the MLL complex in castration-resistant prostate cancer |
|
