|
| Status |
Public on Jan 03, 2015 |
| Title |
HA22T siControl-2 |
| Sample type |
RNA |
| |
|
| Source name |
HA22T siControl-2
|
| Organism |
Homo sapiens |
| Characteristics |
hepatocellular carcinoma cell line: HA22T
transfectant: Control siRNA
|
| Treatment protocol |
HA22T and PLC5 cells were transfected with 10 nM non-targeting and GALNT1 specific siRNA (Thermo Scientific, ON-TARGET plus SMART pool L-011280-01-0005) with Lipofectamine RNAiMAX (Invitrogen) for 48 hours and extracted for total RNA
|
| Growth protocol |
HA22T and PLC5 were cultured in DMEM supplemented with 10% FBS and 0.1 % penicillin and streptomycin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the GeneJET RNA Purification kit (Thermo Scientific, Catelog number: K0731) following the manufacturer's recommendations. RNA was quantified using a NanoDrop spectrophotometer (Bio-Rad) and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA)
|
| Label |
Alexa/CyDye
|
| Label protocol |
cDNA prepared from 10μg of total RNA were labeled with aa-dUTP using Invitrogen SuperScriptTM Plus Indirect cDNA Labeling System according to the manufacturer's protocol, followed by aa-cDNA column purification (QIAGEN, Valencia, CA). Alexa/CyDye was incorporated to aa-cDNA followed by column purification with Alexa/CyDye-cDNA cRNA purification (Qiagen).
|
| |
|
| Hybridization protocol |
Agilent Gene Expression Hybridization Kits was used for hybridization according to the manufacturer’s instruction. Briefly, 16 ul of dye labeled cDNA in water was fragmented at 98°C for 3 mins in a reaction volume of 40 ul containing 4 ul Agilent 10x blocking agenr and 20 ul of Agilent 2xGExHybridization Buffer HI-RPM and hybridized to Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarray (G4851B) at 65°C and rotated at 10 rpm for 17 hours. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (US9230696) using one color scan setting for 8x60k array slides (Scan Area 61x21.6mm, Scan resolution 2um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after 48 hours transfection
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1_105_Dec08 and Grid: 039494_D_F_20120628) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jan 02, 2015 |
| Last update date |
Jan 03, 2015 |
| Contact name |
Min-Chuan Huang |
| E-mail(s) |
mchuang@ntu.edu.tw
|
| Phone |
886-223123456-88177
|
| Organization name |
National Taiwan University
|
| Department |
Graduate Institute of Anatomy and Cell Biology
|
| Street address |
No. 1, Sec. 1, Ren-Ai RD, Taipei, Taiwan, R.O.C.
|
| City |
Taipei |
| ZIP/Postal code |
100 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE64628 |
siRNA knockdown of GALNT1 in hepatocellular carcinoma HA22T and PLC5 cells |
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