Total RNA was extracted by use of the RNeasy Micro Kit (Qiagen).
Label
AlexaFluor555
Label protocol
40 to 50 ng of total RNA were used to prepare aminoallyl-UTP-modified (aaUTP) cRNA (Amino Allyl MessageAmp™ II Kit; #AM1753; Life Technologies) as directed by the company (applying one-round of amplification), except that reaction volumes were halved. The labeling of aaUTP-cRNA was performed by use of Alexa Fluor 555 Reactive Dye (#A32756; LifeTechnologies). Prior to the reverse transcription reaction, 1µl of a 1:100000 dilution of Agilent’s ‘One-Color spike-in Kit stock solution’ (#5188-5282, Agilent Technologies) was added to a subset of total RNA sample.
Hybridization protocol
cRNA fragmentation, hybridization and washing steps were carried-out as recommended in the ‘One-Color Microarray-Based Gene Expression Analysis Protocol V5.7’, except that 300ng of each fluorescently labeled cRNA population were used for hybridization.
Scan protocol
Slides were scanned on the Agilent Micro Array Scanner G2565CA (pixel resolution 3 µm, bit depth 20).
Data processing
Data extraction was performed with the ‘Feature Extraction Software V10.7.3.1’ using the extraction protocol file ‘GE1_107_Sep09.xml’. Measurements of on-chip replicates (quadruplicates) were averaged using the geometric mean of processed intensity values of the green channel, ‘gProcessedSignal’ (gPS) to retrieve one resulting value per probe and sample. 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 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 this latter approach, all 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 normalized (‘Array I’ in the formula shown below). Accordingly, normalized gPS values for all samples (microarray data sets) were calculated by the following formula: normalized gPSArray i = gPSArray i x (1500 / 75th PercentileArray i) A lower intensity threshold (surrogate value) was defined based on intensity distribution of negative control features. This value was fixed to 20. All of those normalized gPS values that fell below this intensity border were substituted by the respective surrogate value of 20.
Shared and unique features distinguishing follicular T helper (TFH) and regulatory (TFR) cells from peripheral lypmh node and Peyer's Patches [Agilent-048306]
Shared and unique features distinguishing follicular T helper (TFH) and regulatory (TFR) cells from peripheral lypmh node and Peyer's Patches
Data table header descriptions
ID_REF
VALUE
Normalized processed signal intensity values (non-log), averaged across on-chip replicates / quadruplicate measurements. Measurements of control features were removed.
