Comment[GEAAccession]	E-GEAD-330
MAGE-TAB Version	1.1
Investigation Title	Knockdown of hSNF5/Ini1 causes call-cycle arrest and apoptosis in a p53-dependent manner
Experiment Description	hSNF5/Ini1 is a component of SWI/SNF complex and has been implicated in tumor suppression. However, knock-out of hSNF5/Ini1 causes cell death by apoptosis. To identify the target gene of hSNF5/Ini1, gene profiling using microarray in conjunction with RNA interference has been employed.
Experimental Design	cellular modification design	time series design
Experimental Factor Name	RNA interference
Experimental Factor Type	RNA interference
Person Last Name	Kato	Watanabe
Person First Name	Hiroyuki	Shinya
Person Affiliation	Department of Clinical Informatics, Tokyo Medical and Dental University	
Person Roles	submitter	submitter
Public Release Date	2019-09-27
PubMed ID	17669367
Protocol Name	P-GEAD-287	P-GEAD-288	P-GEAD-289	P-GEAD-290	P-GEAD-291	P-GEAD-292	P-GEAD-293	P-GEAD-294
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning and feature extraction protocol	normalization data transformation protocol	growth protocol	treatment protocol
Protocol Description	HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FCS) and cultured in DMEM supplemented with 2% FCS after siRNA transfection.	The medium was removed and 10 ml ISOGEN (NIPPON GENE) was immediately pipetted into a 15-cm dish. To prepare total RNA, lysates were further processed according to the manufacturer's instructions. Poly (A)+ RNA was prepared by using the MicroPoly(A)Purist kit (Ambion, Carlsbad, CA, USA).	Poly (A)+ RNA (2microg) was labeled with SuperScript II (Invitrogen) and Cyanine 5-dUTP or Cyanine 3-dUTP (PerkinElmer, MA, USA). Labeling, hybridization, and subsequent washes of microarrays were performed with a Labeling & Hybridization Kit (MicroDiagnostic), in accordance with the manufacturer's instructions.	Labeling, hybridization, and subsequent washes of microarrays were performed with a Labeling & Hybridization Kit (MicroDiagnostic), in accordance with the manufacturer's instructions.	Hybridization signals were measured with a GenePix 4000A scanner (Axon Instruments, CA, USA) and then processed into primary expression ratios ([Cyanine 5-intensity obtained from each target or control sample]/[Cyanine 3-intensity obtained from each control or target sample], which are indicated as 'median of ratios' in GenePix Pro 3.0 software (Axon Instruments)).	Normalization was performed for the median of ratios (primary expression ratios) by multiplying normalization factors calculated for each feature on a microarray by the GenePix Pro 3.0 software (designated normalized ratios). Data processing and subsequent hierarchical clustering analysis were performed with an Excel program (Microsoft, WA, USA) and an MDI gene expression analysis software package (MicroDiagnostic). The primary expression ratios were converted into log2 values (log2 Cyanine 5- intensity/Cyanine 3- intensity)(designated log ratios). After conversion of the normalized ratios into log2 values, log ratios derived from pairs of Cyanine 3-labeled target and Cyanine 5-labeled control samples were converted into reciprocals. Eventually, log ratios derived from pairs of Cyanine 5-labeled target and Cyanine 3-labeled control samples and the reciprocal log ratios derived from pairs of Cyanine 3-labeled target and Cyanine 5-labeled control samples were subjected to calculation of mean averages for individual genes (designated mean log ratios).	HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FCS) and cultured in DMEM supplemented with 2% FCS after siRNA transfection.	siRNAs of the following sequences containing two deoxythymidine residues on the 3' ends of both strands were used. si-hSNF5-sense (+606 to +626 in numbering A of the initiation codon as +1): GUUGAUGACGCCUGAGAUGTT;  si-hSNF5-antisense: CAUCUCAGGCGUCAUCAACTT;  si-GFP-sense (+100 to +120): GGAGUUGUCCCAAUUCUUGTT;  si-GFP-antisense: CAAGAAUUGGGACAACUCCTT. The double-stranded siRNAs were prepared by annealing both strands after EtOH precipitation. Transfection of siRNAs was performed as follows. Approximately 5 106 HeLa cells were plated in 15-cm dish 12-15 h before transfection and 1.2 nmol (usually 30microl of 40microM solution) of double-stranded siRNA was transfected after mixing with 30microl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in 10 ml OPTI-MEM1 (Invitrogen) according to the manufacturer's instruction. Four hours later, the transfection cocktail was removed and DMEM containing 2% fetal bovine serum was supplied to the culture cells in a CO2 incubator. This transfection process was repeated every second day. For microarray profiling, cells were harvested 48 h after the last transfection.
SDRF File	E-GEAD-330.sdrf.txt
Comment[Number of channel]	dual-channel
Comment[Array Design REF]	A-GEAD-8
Comment[AEExperimentType]	transcription profiling by array
Comment[SecondaryAccession]	CBX8
Comment[BioProject]	PRJDB8822
Comment[CIBEX Accept Date]	2005-08-12
Comment[CIBEX Public Release Date]	2007-07-25
Comment[Last Update Date]	2019-09-27