|
| Status |
Public on May 22, 2013 |
| Title |
5H rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HaCaT cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: immortalized human keratinocytes
cell line: HaCaT
treatment: untreated
|
| Treatment protocol |
For treatments, HaCaT cells were serum starved for 24 hr and treated with 5 ug/ ml areca nut water extracts, 5ng/ml TGF-beta (R&D systems, USA) for 48. For treatment with ALK5 inhibitor (TbetaRI inhibitor, Sigma-Aldrich, SB 431542), cells were pre-treated with 10uM of SB 431542 for 2 hr prior to the addition of the respective factors
|
| Growth protocol |
human keratinocytes (HaCaT) were maintained in DMEM (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (Certified grade, Invitrogen corporation, USA. Heat inactivated for HaCaT cells), 100 units/mL penicillin and 100ug/mL streptomycin (Invitrogen Life Sciences, USA) at 37°C in a humidified chamber with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
200 ng of RNA each from untreated and treated samples were used. Labeling of the probes was done using the Low RNA Input Linear Amplification Kit (Agilent Technologies, USA). Briefly 200 ng of RNA were reverse transcribed using T7promoter attached Oligo-dT primers at 40 °C for 2 hrs followed by 65 °C for 15 min. This cDNA were transcribed using T7 polymerase with respective labelled nucleotides at 40 °C for 2hrs. This synthesized labelled cRNA were purified using RNeasy mini column.
|
| |
|
| Channel 2 |
| Source name |
HaCaT cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: immortalized human keratinocytes
cell line: HaCaT
treatment: 5H (5 ug/ml areca nut water extract)
|
| Treatment protocol |
For treatments, HaCaT cells were serum starved for 24 hr and treated with 5 ug/ ml areca nut water extracts, 5ng/ml TGF-beta (R&D systems, USA) for 48. For treatment with ALK5 inhibitor (TbetaRI inhibitor, Sigma-Aldrich, SB 431542), cells were pre-treated with 10uM of SB 431542 for 2 hr prior to the addition of the respective factors
|
| Growth protocol |
human keratinocytes (HaCaT) were maintained in DMEM (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (Certified grade, Invitrogen corporation, USA. Heat inactivated for HaCaT cells), 100 units/mL penicillin and 100ug/mL streptomycin (Invitrogen Life Sciences, USA) at 37°C in a humidified chamber with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
200 ng of RNA each from untreated and treated samples were used. Labeling of the probes was done using the Low RNA Input Linear Amplification Kit (Agilent Technologies, USA). Briefly 200 ng of RNA were reverse transcribed using T7promoter attached Oligo-dT primers at 40 °C for 2 hrs followed by 65 °C for 15 min. This cDNA were transcribed using T7 polymerase with respective labelled nucleotides at 40 °C for 2hrs. This synthesized labelled cRNA were purified using RNeasy mini column.
|
| |
|
| |
| Hybridization protocol |
750 ng of labelled cRNA each from treated and untreated were mixed with blocking agent and fragmentation buffer followd by incubation at 65 °C for 30 min. This is followed by addition of 55 ul of 2x GEx Hybridization Buffer HI-RPM to stop the fragmentation reaction. Later 100 ul of the above mix were added to each array.
|
| Scan protocol |
standard Agilent protocol
|
| Data processing |
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and LOWESS normalization.
|
| |
|
| Submission date |
May 24, 2012 |
| Last update date |
May 23, 2013 |
| Contact name |
Neeraj KUMAR |
| E-mail(s) |
[email protected], [email protected]
|
| Organization name |
Indian Institute of Science Bangalore
|
| Department |
MRDG
|
| Lab |
Prof. P Kondaiah lab
|
| Street address |
Indian Institute of Science Bangalore
|
| City |
Bangalore |
| ZIP/Postal code |
560 012 |
| Country |
India |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE38227 |
Activation of TGF-beta pathway by areca nut constituents: A possible cause of Oral submucous fibrosis. |
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