|
| Status |
Public on Sep 18, 2017 |
| Title |
~E7.75 Noto GFP/+ distal halves technical replicate2 |
| Sample type |
RNA |
| |
|
| Source name |
ventral node-containing distal halves of Noto GFP/+ heterozygotes at the early headfold stage (~E7.75)
|
| Organism |
Mus musculus |
| Characteristics |
strain: NotoGFP, Sv129/CD-1 hybrid background
|
| Treatment protocol |
Noto GFP heterozygous and homozygous embryos were harvested at early headfold stage (~E7.75). The distal third of each embryo comprising the embryonic ventral node at the distal tip was sectioned and collected and pooled in RNA stabilisation solution (RNAlater, Ambion) at 4 °C in pools of 120 to 150 embryonic thirds.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Micro or Mini Kit (Qiagen) following the manufacturer’s instructions (including DNase digest). Embryos were homogenised with a micro pestle in a 1.5 ml Eppendorf tube. Total RNA was eluted in 14-15 µl H2O resulting in an RNA concentration of around 400 ng/µl.
|
| Label |
Cy5
|
| Label protocol |
Synthesis of Cy3- or Cy5-labelled cRNA was performed with the ‘Quick Amp Labeling kit, two color’ (#5190-0444, Agilent Technologies) according to the manufacturer’s recommendations using 500-1000 ng of total RNA.
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| |
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| Hybridization protocol |
cRNA fragmentation, hybridisation and washing steps were carried out as recommended in the ‘Two-Color Microarray-Based Gene Expression Analysis Protocol V5.7’ (Agilent), except that 4000-5000 ng of each labelled cRNA population were used for hybridisation.
|
| Scan protocol |
Slides were scanned using the Agilent Micro Array Scanner G2565BA at two different photomultiplier tube (PMT) settings (100 % and 5 %) to increase the dynamic range of the measurement (extended dynamic range mode).
|
| Data processing |
Data extraction and processing of raw fluorescence intensity values were performed with the ‘Feature Extraction software V9.5.3.1’ by using the extraction protocol file: GE2-v5_95_Feb07.xml. Measurements of on-chip replicated features were averaged using the geometric mean of processed intensity values from both channels, ‘gProcessedSignal’ (gPS) and ‘rProcessedSignal’ (rPS), separately to retrieve one resulting value per unique non-control probe. Single features were excluded from averaging if they i) were manually flagged, ii) were identified as outliers by the Feature Extraction software, iii) lay outside the interval of ‘1.42 x interquartile range‘ regarding the PS distribution of the respective on-chip replicate population, or iv) showed a coefficient of variation of pixel intensities per feature that exceeded 0.5. For inter-array normalisation, PS values were further transformed as follows: All PS values (gPS and rPS) of one particular microarray (‘Array i’ in the formula shown below) were multiplied with a microarray-specific scaling factor. This factor was calculated by dividing a ‘reference 75th percentile value’ (targeted at 1500 for the whole series) by the 75th percentile of the gPS values calculated by the Feature Extraction software for that microarray. Accordingly, normalised PS values (nPS) for all samples (microarray data sets) were calculated by the following formula: normalised PSArray i = PSArray i x (1500 / 75th Percentile gPSArray i) Finally, a lower intensity threshold was defined as 1% of the reference 75th percentile value (= 15). All nPS values below this intensity cutoff were substituted by the surrogate value of 15.
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| |
|
| Submission date |
Nov 06, 2016 |
| Last update date |
Sep 18, 2017 |
| Contact name |
Oliver Dittrich-Breiholz |
| E-mail(s) |
dittrich.oliver@mh-hannover.de
|
| Organization name |
Medical School Hannover
|
| Department |
Research Core Unit Genomics
|
| Street address |
Carl-Neuberg-Str. 1
|
| City |
Hannover |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE89583 |
Identification of FOXJ1 effectors during ciliogenesis in the fetal respiratory epithelium and embryonic left-right organizer of the mouse - Noto screen |
| GSE89717 |
Identification of FOXJ1 effectors during ciliogenesis in the fetal respiratory epithelium and embryonic left-right organizer of the mouse |
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