|
| Status |
Public on Nov 16, 2018 |
| Title |
M5339: E13.5 ureters, Tbx2-GOF vs control, rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
control
|
| Organism |
Mus musculus |
| Characteristics |
strain: NMRI
genotype: Tbx18(+/+);Hprt(Tbx2/Y)
gender: male
developmental stage: E13.5
tissue: Ureter
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using peqGOLD RNAPure™ (VWR) 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.
|
| |
|
| Channel 2 |
| Source name |
Tbx2-GOF
|
| Organism |
Mus musculus |
| Characteristics |
strain: NMRI
genotype: Tbx18(cre/+);Hprt(Tbx2/Y)
gender: male
developmental stage: E13.5
tissue: Ureter
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using peqGOLD RNAPure™ (VWR) 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.
|
| |
|
| |
| Hybridization protocol |
cRNA fragmentation, hybridization and washing steps were carried-out exactly as recommended in the 'Two-Color Microarray-Based Gene Expression Analysis Protocol V5.7' (Agilent Technologies), except that 2500ng of each labeled cRNA population were used for hybridization.
|
| Scan protocol |
Slides were scanned on the Agilent Micro Array Scanner G2565CA (pixel resolution 5 µm, bit depth 20).
|
| 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 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) by the 75th Percentile of the microarray dataset to be normalized. 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 15 normalized PS units. All of those measurements that fell below this intensity cutoff were substituted by the respective surrogate value of 15.
|
| |
|
| Submission date |
Nov 15, 2018 |
| Last update date |
Nov 16, 2018 |
| Contact name |
Andreas Kispert |
| E-mail(s) |
kispert.andreas@mh-hannover.de
|
| Phone |
0049 5115324017
|
| Organization name |
Medizinische Hochschule Hannover
|
| Department |
Molekularbiologie
|
| Lab |
Kispert
|
| Street address |
Carl-Neuberg-Str. 1
|
| City |
Hannover |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL11202 |
| Series (2) |
| GSE122558 |
Transcripts identified by microarray analysis that were deregulated in E13.5 Tbx18cre/+;HprtTbx2/Y (Tbx2-GOF) ureters |
| GSE122561 |
TBX2 and TBX3 act downstream of canonical WNT signaling in patterning and differentiation of the ureteric mesenchyme |
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