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| Status |
Public on May 22, 2013 |
| Title |
R2 |
| Sample type |
RNA |
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| Source name |
Serum
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| Organism |
Homo sapiens |
| Characteristics |
group: Rapid biochemical recurrence
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from serum essentially as described previously (Selth et al, Int J Cancer, 131, 652–661, 2012), with some minor modifications. Serum was centrifuged at 3,000g for 5 min at 4°C prior to extraction. A serum:Qiazol ratio of 1:5 was used to denature the samples. miRNAs were extracted from 500 ml serum spiked with 25 fmol synthetic cel-miR-39 (Integrated DNA Technologies) and eluted in 35 ml RNase-free water.
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| Label |
SYBR Green
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| Label protocol |
PCR assays were performed using theTaqMan Array Human MicroRNA A+B Cards Set v2.0 (Applied Biosystems), as per the manufacturer’s instructions. We used 3ul of serum miRNA as input for the Megaplex reverse transcription (RT) reactions. After RT and pre-amplification, quantitative PCR was carried out on an Applied Biosystems 7900HT.
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| Hybridization protocol |
n/a
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| Scan protocol |
n/a
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| Description |
SAMPLE 2
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| Data processing |
Cycle threshold values were calculated using SDS 2.4 software with manual baseline (3-15) and threshold (0.2) settings. Undetected miRNAs (average Ct > 35 in either the recurrence or no recurrence groups) were removed from the analysis. Normalisation of the data was as follows: 1) the A plates and B plates were normalised to each other using the Ct values for U6 snRNA, which is amplified on both plates; 2) all data was then scaled to the spiked-in cel-miR-39 to correct for differences in extraction efficiency, 3) finally, the mean Ct value of all miRNAs with a Ct value < 30 for each array was determined and used to normalise the arrays.
Matrix normalized worksheet reports normalized signal (described above).
Fold Change worksheet reports recurrence/no recurrence ratios.
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| Submission date |
May 21, 2013 |
| Last update date |
May 22, 2013 |
| Contact name |
Luke Selth |
| E-mail(s) |
[email protected]
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| Organization name |
Flinders University
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| Department |
College of Medicine and Public Health
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| Street address |
Flinders Drive
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| City |
Bedford Park |
| State/province |
SA |
| ZIP/Postal code |
5042 |
| Country |
Australia |
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| Platform ID |
GPL16850 |
| Series (1) |
| GSE47125 |
Circulating microRNAs predict biochemical recurrence in prostate cancer patients |
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| Supplementary data files not provided |
| Processed data are available on Series record |
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