|
| Status |
Public on Feb 20, 2016 |
| Title |
LP-2 [mouse] |
| Sample type |
RNA |
| |
|
| Source name |
mammary gland
|
| Organism |
Mus musculus |
| Characteristics |
strain: FVB/N
cell type: Luminal progenitor (LP)
mouse pool: 2
|
| Treatment protocol |
no treatment
|
| Growth protocol |
Cells were freshly isolated from adult mouse mammary glands for microRNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
microRNA was extracted with the Qiagen miRNAeasy kit
|
| Label |
FAM-MGB
|
| Label protocol |
Complementary cDNA was prepared using TaqMan microRNA Reverse Transcription Kit (ABI 4366596) and the Megaplex RT primer pools (mouse v2.0 ABI #4401012) according to the manufacturer’s protocol. For each sample, cDNA was made using appropriate Megaplex primer pools and 3 µl of RNA as template. Each pool was pre-amplified for 12 cycles using TaqMan PreAmp Master Mix (ABI #4391128) and Megaplex preAmp primers (human v2.0 ABI #4401091). Quantitative RT-PCR was carried out on an ABI 7900HT machine using TaqMan Rodent MicroRNA Array Set v2.0 (ABI #4400239) Taqman Low Density Array plates and Universal PCR Master Mix (ABI 4364341) according to the manufacturer’s protocol. U6 was included as an endogenous control and Arabidopsis thaliana-miR159a was included as a negative control on each plate.
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
Sample 6
|
| Data processing |
Ct values were exported using Applied Biosystems's SDS v2.3 and RQ Manager v1.2 software, with automatic baseline and a manual Ct threshold of 0.2. Normalization and statistical analysis used the limma software package. Ct values were transformed to a log2 expression scale by subtracting the Ct values from 40.5. The log2 expression values were then normalized by cyclic loess normalization with the MammU6 house-keeping probe up-weighted 100-fold. The cyclic method was set to “affy”, the loess span was 0.7 and 5 cyclic iterations were used. After normalization, probes were filtered out as unexpressed if they failed to achieve a normalized value of 2 in at least 3 samples. Differential expression analysis used linear models including a batch correction for mouse pool. Statistical significance was asssessed using empirical Bayes t-statistics.
Normalized worksheet reports limma normalized log2 expression signals
Fold change worksheet reports a limma topTable giving differentially expressed miRNAs in the MaSC population vs the average of the LP and ML populations.
|
| |
|
| Submission date |
Mar 19, 2015 |
| Last update date |
Feb 20, 2016 |
| Contact name |
Gordon K Smyth |
| E-mail(s) |
smyth@wehi.edu.au
|
| Phone |
(+61 3) 9345 2326
|
| Fax |
(+61 3) 9347 0852
|
| URL |
http://www.wehi.edu.au
|
| Organization name |
Walter and Eliza Hall Institute of Medical Research
|
| Department |
Bioinformatics
|
| Lab |
Smyth
|
| Street address |
1G Royal Pde
|
| City |
Parkville |
| State/province |
Vic |
| ZIP/Postal code |
3052 |
| Country |
Australia |
| |
|
| Platform ID |
GPL10331 |
| Series (2) |
| GSE67055 |
microRNA expression profiles of distinct mouse mammary epithelial cell types |
| GSE67056 |
microRNA expression profiles of distinct human and mouse mammary epithelial cell types |
|
