|
| Status |
Public on Feb 26, 2015 |
| Title |
Control day 4, rep 3 |
| Sample type |
RNA |
| |
|
| Source name |
HMLE, empty control vector, day 4
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HMLE
stably expressing: empty vector
treatment: 4-OHT
time point: day 4
|
| Treatment protocol |
Cells were treated with fresh media containing 20nM 4-OHT every 2 days.
|
| Growth protocol |
Cells were maintained in MEGM media.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using miRNeasy Micro (Qiagen) according to manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Microarray assay was performed using a service provider (LC Sciences). The assay started from 4 to 8 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore) and the small RNAs (< 300 nt) isolated were 3'-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent Cy3/Cy5 dye staining.
|
| |
|
| Hybridization protocol |
Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://microrna.sanger.ac.uk/sequences/) or other RNA (control or customer-defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 uL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C.
|
| Scan protocol |
Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
|
| Description |
V d4-3
Biological replicate 3 of 3. HMLE cells stably expressing a control empty vector, treated 4 days with 4-OHT.
|
| Data processing |
Background is determined using a regression-based background mapping method. The regression is performed on 5% to 25% of the lowest intensity data points excluding blank spots. Raw data matrix is then subtracted by the background matrix.
Normalization is carried out using a LOWESS (Locally-weighted Regression) method on the background-subtracted data. The normalization is to remove system-related variations, such as sample amount variations, different labeling dyes, and signal gain differences of scanners so that biological variations can be faithfully revealed [B. M. Bolstad, R. A. Irizarry, M. Astrandand T. P. Speed, (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias, Bioinformatics, 19 (2), 185-193].
A transcript to be listed as detectable must meet at least two conditions: signal intensity higher than 3×(background standard deviation) and spot CV < 0.5. CV is calculated by (standard deviation)/(signal intensity). When repeating probes are present on an array, a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level.
t-Test is performed between control and test sample groups. T-values are calculated for each miRNA, and p-values are computed from the theoretical t-distribution. miRNAs with p-values below a critical p-value (typically 0.01) are selected for cluster analysis. The clustering is done using hierarchical method and is performed with average linkage and Euclidean distance metric.
All data processes, except clustering plot, are carried out using in-house developed computer programs. The clustering plot is generated using TIGR MeV (Multiple Experimental Viewer) software from The Institute for Genomic Research.
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| |
|
| Submission date |
Jun 16, 2014 |
| Last update date |
Feb 27, 2015 |
| Contact name |
David Drasin |
| Organization name |
University of Colorado AMC
|
| Department |
Pharmacology
|
| Lab |
Heide Ford
|
| Street address |
12800 E. 19th Ave, MS 8303
|
| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL18795 |
| Series (1) |
| GSE58560 |
TWIST1-induced microRNA-424 drives an intermediate epithelial-to-mesenchymal transition that opposes metastasis |
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