CD45high CD11bhigh (CD45R/B220, CD90.2/Thy-1.2, NK‑1.1, CD49b/DX5, Ly6G)low myeloid cells were isolated by FACS from the infarcted left ventricle and spleen 3 days after AMI in C57BL/6N wild-type mice.
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified using the PicoPure kit (Thermo Fisher Scientific; catalogue number, KIT0204).
Label
Alexa Fluor 555
Label protocol
Aminoallyl-UTP (aa-UTP)-modified cRNA was prepared from 10 ng total RNA using the Amino Allyl MessageAmp II kit (Thermo Fisher Scientific; catalogue number, AM1753) and applying one round of amplification as recommended by the company; aa-UTP-cRNA was labeled with Alexa Fluor 555 reactive dye (Thermo Fisher Scientific; catalogue number, A32756) as described in the Amino Allyl MessageAmp II kit. All reaction volumes were down-scaled by a factor of 2.
Hybridization protocol
cRNA fragmentation, hybridization to Whole Mouse Genome Oligo Microarrays V2 (Agilent Technologies, Agilent MicroArray Design Identifier 026655), and washing were performed as recommended in the One-Color Microarray-Based Gene Expression Analysis Protocol V5.7, except that 300 ng of each fluorescently labeled cRNA population were used for hybridization.
Scan protocol
Slides were scanned with an Agilent G2565CA microarray scanner system (pixel resolution 5 µm, bit depth 20).
Description
Myeloid cells from the infarctd left ventricle
Data processing
Data were extracted with Feature Extraction Software V10.7.3.1 using the extraction protocol file GE1_107_Sep09.xml. Measurements of on-chip replicates were averaged using the geometric mean of processed intensity values of the green channel, ‘gProcessedSignal’ (gPS), to retrieve one resulting value per unique non-control probe. Single features were excluded from averaging, if they were (i) manually flagged, (ii) were identified as outliers by the Feature Extraction Software, (iii) lay outside the interval of ‘1.42x interquartile range‘ regarding the normalized gPS distribution of the respective on-chip replicate population, or (iv) showed a coefficient of variation of pixel intensities per feature that exceeded 0.5. Averaged gPS values were normalized by Quantile normalization first, followed by global linear scaling. For the latter approach all quantile normalized gPS values of one sample were multiplied by an array-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 particular Microarray to be finally normalized (‘Array I’ in the formula shown below). Accordingly, finally normalized gPS values for all samples (microarray data sets) were calculated by the following formula: finally normalized gPSArray i = quantile normalized gPSArray i x (1500 / 75th PercentileArray i) Finally, a lower intensity threshold (surrogate value) was defined based on intensity distribution of negative control features. This value was fixed at 15 finally normalized gPS units. All of those measurements that fell below this intensity cutoff were substituted by the respective surrogate value of 15. Finally, normalized processed data were imported into Qlucore Omics Explorer software under default import settings for Agilent One Color mRNA microarrays, except that any (further) normalization option during import was deselected. Accordingly, data processing steps during Omics Explorer import were (i) removal of control measurements, (ii) log2 transformation, and (iii) baseline transformation to the median.
Submission date
Mar 10, 2022
Last update date
Jun 20, 2022
Contact name
Marc R. Reboll
Organization name
Hannover Medical School
Department
Molecular and Translational Cardiology, Department of Cardiology and Angiology
Transcriptome analysis of myeloid cells after acute myocardial infarction (AMI) in C57BL/6N wild-type mice.
Data table header descriptions
ID_REF
VALUE
Normalized processed signal intensity values, averaged across on-chip replicates / quintuplicate measurements, log2 and baseline transformed. Measurements of control features were removed.
