|
| Status |
Public on May 25, 2016 |
| Title |
HT shOCT2_1196 DOX Treated - 2 days - mAdbID:118327 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HT shOCT2_1196 DOX Treated - 2 days
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HT
cell type: GCB DLBCL cells
disease state: germinal center B cell-restricted (GCB) subtype of diffuse large B cell lymphoma (DLBCL)
|
| Treatment protocol |
Treatment type: shRNA transduction
Treatment dose: 50 ng/mL
Treatment time: 2 days
In-vitro treatment: Cells were transduced with retroviral vectors expressing shOCT2_1196. shRNA expression was induced with 50 ng/mL doxycycline for 2 days.
Other: Plasmids: OCT2 was cloned from cDNA from BJAB and OCA-B cDNA purchased from Origene. Both were cloned into the vectors pCMV-TOP and pFlagBiopCMV-TOP(30). Site directed mutations were introduced using the QuikChange Lightning kit from Agilent. shRNAs targeting OCT2 or OCA-B were designed and cloned as double stranded oligonucleotides into a retroviral vector (pRSMX_PuroGFP) for doxycycline-inducible shRNA expression. RNAi sequences used are as follows: OCT2 shRNA#1_1196 5'-GCACAACAGTTACTACCTTAT-3', OCT2 shRNA#2_3410 5'-GGATGCTTCTTTCTCTTCACA-3', OCA-B shRNA_3017 5'-CAGCCAGAAGTACCATTAGG-3'. Because OCT2 shRNA#1 targeted the coding region of OCT2, silent mutations were introduced into the recognition site to allow the shRNA to be rescued by OCT2 constructs. Vectors: The pRSMX-PG vector coexpressing GFP and shRNA was introduced into cells and where required selection was carried out with puromycin. Expression of shRNA was induced by addition of doxycycline (50 ng/mL).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TRIzol Extraction Protocol
Other: Total RNA was prepared by the TRIzol method (Invitrogen) and purified using RNeasy Mini columns (Qiagen).
|
| Label |
cy3
|
| Label protocol |
Agilent Labeling-Cy3
Other: Total RNA was reverse transcribed to cDNA using T7 Promoter Primer and MMLV-RT. Then the cDNA was converted to aRNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP (Two-Color Microarray-Based Gene Expression Analysis Protocol, Agilent).
|
| |
|
| Channel 2 |
| Source name |
HT shControl DOX Treated - 2 days
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HT
cell type: GCB DLBCL cells
disease state: germinal center B cell-restricted (GCB) subtype of diffuse large B cell lymphoma (DLBCL)
|
| Treatment protocol |
Treatment type: shRNA transduction
Treatment dose: 50 ng/mL
Treatment time: 2 days
In-vitro treatment: Cells were transduced with retroviral vectors expressing shControl. shRNA expression was induced with 50 ng/mL doxycycline for 2 days.
Other: Plasmids: OCT2 was cloned from cDNA from BJAB and OCA-B cDNA purchased from Origene. Both were cloned into the vectors pCMV-TOP and pFlagBiopCMV-TOP(30). Site directed mutations were introduced using the QuikChange Lightning kit from Agilent. shRNAs targeting OCT2 or OCA-B were designed and cloned as double stranded oligonucleotides into a retroviral vector (pRSMX_PuroGFP) for doxycycline-inducible shRNA expression. RNAi sequences used are as follows: OCT2 shRNA#1_1196 5'-GCACAACAGTTACTACCTTAT-3', OCT2 shRNA#2_3410 5'-GGATGCTTCTTTCTCTTCACA-3', OCA-B shRNA_3017 5'-CAGCCAGAAGTACCATTAGG-3'. Because OCT2 shRNA#1 targeted the coding region of OCT2, silent mutations were introduced into the recognition site to allow the shRNA to be rescued by OCT2 constructs. Vectors: The pRSMX-PG vector coexpressing GFP and shRNA was introduced into cells and where required selection was carried out with puromycin. Expression of shRNA was induced by addition of doxycycline (50 ng/mL).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TRIzol Extraction Protocol
Other: Total RNA was prepared by the TRIzol method (Invitrogen) and purified using RNeasy Mini columns (Qiagen).
|
| Label |
cy5
|
| Label protocol |
Agilent Labeling-Cy5
Other: Total RNA was reverse transcribed to cDNA using T7 Promoter Primer and MMLV-RT. Then the cDNA was converted to aRNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP (Two-Color Microarray-Based Gene Expression Analysis Protocol, Agilent).
|
| |
|
| |
| Hybridization protocol |
Agilent Hybridization
Other: According to the manufacture's recommended protocol (Two-Color Microarray-Based Gene Expression Analysis, Agilent).
|
| Scan protocol |
Scan_MicronsPerPixelX: 5
Scan_MicronsPerPixelY: 5
Scan_ScannerName: Agilent Technologies Scanner G2505C US45102888
Agilent Scanning Protocol
Other: Arrays were scanned at 5um resolution on an Agilent DNA Microarray Scanner (G2505C, Agilent) using the default settings for 4x44k format two-color arrays.
|
| Description |
mAdb experiment ID: 118327
|
| Data processing |
Agilent Data Processing Protocol
Calculation Method: Images were auto gridded, analyzed and data extracted using Agilent Feature Extraction Software. Spot values were normalized using the default linear-lowess normalization.
FeatureExtractor_Version: 10.1.1.1
Protocol_Name: GE2-v5_10_Apr08 (Read Only)
|
| |
|
| Submission date |
Mar 16, 2016 |
| Last update date |
May 25, 2016 |
| Contact name |
Louis M. Staudt |
| E-mail(s) |
lstaudt@mail.nih.gov
|
| Phone |
301-402-1892
|
| Organization name |
National Cancer Institute
|
| Department |
Lymphoid Malignancies Branch
|
| Lab |
Louis M Staudt
|
| Street address |
9000 Rockville Pike, Bldg 10, Rm 4N114
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL4133 |
| Series (2) |
| GSE79292 |
Regulation of normal B cell differentiation and malignant B cell survival by OCT2 (expression) |
| GSE79482 |
Regulation of normal B cell differentiation and malignant B cell survival by OCT2 |
|
