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Status |
Public on Jan 04, 2022 |
Title |
E18.5 ureters, Rbpj-cKO vs control, rep2 |
Sample type |
RNA |
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Channel 1 |
Source name |
control
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Organism |
Mus musculus |
Characteristics |
strain: NMRI genotype: Rbpj(fl/?) gender: female developmental stage: E18.5 tissue: ureter
|
Treatment protocol |
Ureters were mechanically dissected from E18.5 embryos.
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Extracted molecule |
total RNA |
Extraction protocol |
Extraction of total RNA was performed using peqGOLD RNAPure (VWR Peqlab) according to the manufacturer’s recommendations.
|
Label |
AlexaFluor647
|
Label protocol |
Synthesis of AlexaFluor555- or AlexaFluor647- (Invitrogen) labeled cRNA was performed with the ‘Amino Allyl MessageAmp™ II aRNA Amplification Kit’ (#AM1753, Life Technologies) according to the manufacturer’s recommendations, except that the proportion of aminoallyl-UTP/UTP was adjusted to 1+11 (instead of 1+1) and that reaction volumes were halved.
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Channel 2 |
Source name |
Rbpj-cKO
|
Organism |
Mus musculus |
Characteristics |
strain: NMRI genotype: Tbx18(cre/+);Rbpj(fl/fl) gender: female developmental stage: E18.5 tissue: ureter
|
Treatment protocol |
Ureters were mechanically dissected from E18.5 embryos.
|
Extracted molecule |
total RNA |
Extraction protocol |
Extraction of total RNA was performed using peqGOLD RNAPure (VWR Peqlab) according to the manufacturer’s recommendations.
|
Label |
AlexaFluor555
|
Label protocol |
Synthesis of AlexaFluor555- or AlexaFluor647- (Invitrogen) labeled cRNA was performed with the ‘Amino Allyl MessageAmp™ II aRNA Amplification Kit’ (#AM1753, Life Technologies) according to the manufacturer’s recommendations, except that the proportion of aminoallyl-UTP/UTP was adjusted to 1+11 (instead of 1+1) and that reaction volumes were halved.
|
|
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Hybridization protocol |
cRNA fragmentation, hybridization and washing steps were carried-out exactly as recommended for 4-plex Microarray-Slides in the ‘Two-Color Microarray-Based Gene Expression Analysis Protocol V5.7’ (Agilent Technologies).
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Scan protocol |
Slides were scanned on the Agilent Micro Array Scanner G2565CA (pixel resolution 5 µm, bit depth 20).
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Data processing |
Data extraction, processing and intra-array normalization of raw fluorescence intensity values were performed with the ‘Feature Extraction Software V10.7.3.1’ by using the recommended default extraction protocol file ‘GE2_107_Sep09.xml’. Measurements of on-chip replicates were averaged using the geometric mean of ProcessedSignal (PS) values (for each channel independently) to retrieve one resulting value per non-control probe, sample, and channel. 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 normalization, averaged processed intensity values, ‘gProcessedSignal’ (gPS) and ‘rProcessedSignal’ (rPS), 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’ (set as 1500 for the whole series) by the 75th Percentile value of the gPS values calculated by the Feature Extraction software for that microarray. Accordingly, normalized PS values for all samples (microarray data sets) were calculated by the following formula: normalized PSArray i = PSArray i x (1500 / 75th Percentile gPSArray i) Finally, a lower intensity threshold (surrogate value) was defined based on intensity distribution of negative control features. This value was fixed at 10 normalized PS units. All of those measurements that fell below this intensity cutoff were substituted by the respective surrogate value of 10.
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Submission date |
Mar 25, 2021 |
Last update date |
Jan 04, 2022 |
Contact name |
Andreas Kispert |
E-mail(s) |
kispert.andreas@mh-hannover.de
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Phone |
0049 5115324017
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Organization name |
Medizinische Hochschule Hannover
|
Department |
Molekularbiologie
|
Lab |
Kispert
|
Street address |
Carl-Neuberg-Str. 1
|
City |
Hannover |
ZIP/Postal code |
30625 |
Country |
Germany |
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Platform ID |
GPL11202 |
Series (1) |
GSE169662 |
Transcripts identified by microarray analysis that were deregulated in E18.5 Tbx18(cre/+);Rbpj(fl/fl) (Rbpj-cKO) ureters |
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