|
| Status |
Public on Feb 02, 2010 |
| Title |
HL60, BR2, 0hr |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HL60 cells not treated with vitamin D
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HL60
vitamin d exposure: 0hrs
|
| Treatment protocol |
We stimulated cells with 0.1 microM calcitriol for 0 hours, 8 hours or 36 hours
|
| Growth protocol |
We used nine cell lines (Hep-G2, HL60, K562, AG09309, AG09319, GM15084, GM12878, GM07348 and GM07019). These were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 0.2 mM L-glutamine at 37 oC in 5% humidified CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After harvesting, cells were lysed in 500 uL of lysis buffer (Promega). Homogenised samples were immediately stored at -80 oC
|
| Label |
Hy3
|
| Label protocol |
400 ng total RNA from sample and reference was labelled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labelling kit (Exiqon, Denmark) following the procedure described by the manufacturer
|
| |
|
| Channel 2 |
| Source name |
Common reference (S1-S54)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Common reference (S1-S54)
vitamin d exposure: --
|
| Treatment protocol |
We stimulated cells with 0.1 microM calcitriol for 0 hours, 8 hours or 36 hours
|
| Growth protocol |
We used nine cell lines (Hep-G2, HL60, K562, AG09309, AG09319, GM15084, GM12878, GM07348 and GM07019). These were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 0.2 mM L-glutamine at 37 oC in 5% humidified CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After harvesting, cells were lysed in 500 uL of lysis buffer (Promega). Homogenised samples were immediately stored at -80 oC
|
| Label |
Hy5
|
| Label protocol |
400 ng total RNA from sample and reference was labelled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labelling kit (Exiqon, Denmark) following the procedure described by the manufacturer
|
| |
|
| |
| Hybridization protocol |
The Hy3™-labeled samples and a Hy5™-labeled reference RNA sample were mixed pair-wise and hybridized to the miRCURY™ LNA array version 11.0 (Exiqon, Denmark), which contains capture probes targeting all miRNAs for human, mouse or rat registered in the miRBASE version 14.0 at the Sanger Institute. The hybridization was performed according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridization station (Tecan, Austria)
|
| Scan protocol |
After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY™ LNA array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene 8.0 software (BioDiscovery, Inc., USA)
|
| Description |
HL60 cells not treated with vitamin D
|
| Data processing |
The quantified signals were background corrected (Normexp with offset value 10 [21]) and normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm
|
| |
|
| Submission date |
Feb 01, 2010 |
| Last update date |
Feb 01, 2010 |
| Contact name |
Adam Handel |
| E-mail(s) |
ahandel@doctors.org.uk
|
| Organization name |
University of Oxford
|
| Department |
Wellcome Trust Centre for Human Genetics
|
| Lab |
Ebers Group
|
| Street address |
Roosevelt Drive
|
| City |
Oxford |
| ZIP/Postal code |
OX3 7BN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL7723 |
| Series (1) |
| GSE20122 |
Vitamin D and microRNA Expression |
|
