|
| Status |
Public on Oct 05, 2016 |
| Title |
Prefrontal cortex_C57Bl6_replicate_1 |
| Sample type |
RNA |
| |
|
| Source name |
C57BL/6J prefrontal cortex
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6J
genotype/variation: wild type
gender: male
age: 5 months
tissue: whole prefrontal cortex
|
| Treatment protocol |
Mice were housed in standard cages and received no pharmacological treatment. Prefrontal cortices were freshly dissected and frozen on dry ice.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNAs from dissected prefrontal cortices (n=3 per experimental group) were purified using standard column purification according to the manufacturer’s protocol (RNAeasy Mini Kit, QIAGEN). RNA quality was analyzed by microfluidic gel electrophoresis on RNA 6000 NanoChips using the Agilent 2100 Bioanalyzer. Only RNA with a high (9) RNA integrity number was selected and used for subsequent retrotranscription, labeling, and array hybridization according to Agilent protocols.
|
| Label |
N/A
|
| Label protocol |
100ng of total RNA was processed by reverse transcriptase and pre-amplification steps following the manufacturer’s protocol (Applied Biosystems). The pre-amplification reaction was mixed with TaqMan OpenArray Real-Time PCR Master mix (1:1). The mix was loaded onto the OpenArray rodent panel (750 mouse/rat miRNAs) using the Accufill System and ran using a QuantStudio 12K Flex PCR (Life Technologies).
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| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
Control
|
| Data processing |
All analyses were performed in R Bioconductor (R Core Team, 2016; Huber et al., 2015). The data was filtered as follows, if the cycle threshold (Ct) score for any miRNA in any sample was was > 35, then the Ct score was set to 40 (i.e. it was considered “Undetermined”). If the Ct was “Undetermined” in 4 or more samples for any miRNA then that miRNA was removed. The data was quantile-normalized and re-filtered by setting any Ct score that was > 30 to 40 (i.e. “Undetermined”). Again, if the Ct was “Undetermined” in 4 or more samples for any miRNA then that miRNA was removed. Using these criteria 306 miRNAs remained for the final data analysis. Differential expression analysis was performed by applying a Student’s t-test to the normalized Ct values between the two conditions and the p-values were adjusted for multiple testing by controlling the false discovery rate (FDR) according to the method of Benjamini and Hochberg (Benjamini and Hochberg, 1995). A miRNA was considered to be differentially expressed if the adjusted p-value was ≤ 0.05. The relative quantification (FC) was calculated as RQ = 2^−∆∆CT .
Fold Change worksheet reports FC, ΔΔCt, mean Control (B6), mean BTBR, t.test, p.value and adj.p.value. FC was calculated as 2^-ΔΔCt, where -ΔΔCt = -[ΔCt_test -Δct_control]. A Student’s t-test was applied to the normalized Ct values between the two conditions and the p-values were adjusted for multiple testing by controlling the false discovery rate (FDR) according to the method of Benjamini and Hochberg (Benjamini and Hochberg, 1995).
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| |
|
| Submission date |
Oct 04, 2016 |
| Last update date |
Oct 05, 2016 |
| Contact name |
David Henshall |
| Organization name |
Royal College of Surgeons in Ireland
|
| Street address |
123 St Stephens Green
|
| City |
Dublin |
| ZIP/Postal code |
2 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL22525 |
| Series (1) |
| GSE87601 |
Prefrontal cortex gene expression signature in autistic BTBR mice |
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