|
| Status |
Public on Jun 19, 2019 |
| Title |
RSBS9 CD133 -ve |
| Sample type |
RNA |
| |
|
| Source name |
Primary cultured cells
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: Cervical cancer
gender: Female
|
| Treatment protocol |
Sorted the cells with Flow cytometry with ALDH and CD133 antibody
|
| Growth protocol |
Cells Cultured in DMEM-F12 media (Invitrogen, USA) and 10%FBS (USA origin), at 37C in 5%CO2 till 70%confluency
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The RNA extraction from the samples was performed by Qiagen RNeasy Mini Kit with DNAse treatment (Cat.No. 74004) as per manufacturer’s protocol.
|
| Label |
Cy4
|
| Label protocol |
The samples for Gene expression were labeled using Agilent Quick-Amp labeling Kit (p/n5190-0442). 500ng each of total RNA were reverse transcribed at 40°C using oligo dT primer tagged to a T7 polymerase promoter and converted to double stranded cDNA. Synthesized double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up using Qiagen RNeasy columns (Qiagen, Cat No: 74106) and quality assessed for yields and specific activity using the Nanodrop ND-1000.
|
| |
|
| Hybridization protocol |
600ng of labeled cRNA sample were fragmented at 60 º C and hybridized on to a Agilent's human GXP_8X60k Array . (AMADID: 039494). Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of (Agilent Technologies, In situ Hybridization kit, Part Number 5190-0404). Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C for 16 hours.
|
| Scan protocol |
Agilent Microarray Scanner (Agilent Technologies, Part Number G2600D).
|
| Description |
Gene expression after sorting cells
|
| Data processing |
Images were quantified using Feature Extraction Software ( Agilent). Feature extracted raw data was analyzed using GeneSpring GX software from Agilent. Normalization of the data was done in GeneSpring GX using the percentile shift normalization method .
|
| |
|
| Submission date |
Sep 20, 2018 |
| Last update date |
Jun 19, 2019 |
| Contact name |
Shifa Javed |
| E-mail(s) |
shifajvd@gmail.com
|
| Organization name |
Postgraduate Institute of Medical Education and Research
|
| Department |
Cytology and GynecPathology
|
| Lab |
Molecular Pathology
|
| Street address |
Sector 12, Chandigarh, India
|
| City |
Chandigarh |
| State/province |
Outside US |
| ZIP/Postal code |
160012 |
| Country |
India |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE120270 |
Gene expression profiling of the cancer stem cells of cervical cancer vs the bulk population |
|
