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Status |
Public on May 25, 2016 |
Title |
BJAB shOCT2_3410 DOX Treated - 2 days - mAdbID:117929 |
Sample type |
RNA |
|
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Channel 1 |
Source name |
BJAB shOCT2_3410 DOX Treated - 2 days
|
Organism |
Homo sapiens |
Characteristics |
cell line: BJAB 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_3410. 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).
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Channel 2 |
Source name |
BJAB shControl DOX Treated - 2 days
|
Organism |
Homo sapiens |
Characteristics |
cell line: BJAB 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).
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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.
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Description |
mAdb experiment ID: 117929
|
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)
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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 |
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