cell type: bronchoalveolar lavage (BAL) cells diagnosis: IPF cohort: LEUVEN age: 58 sex (0=female, 1=male): 1 time to death (days): 362 survival status, 0 = censored, 1 = death: 0 gap: 4
Extracted molecule
total RNA
Extraction protocol
[Isolation protocol] BAL cells were obtained by bronchoscopy, immediately isolated and 1 ml of TRIzol (15596018, life technologies, Darmstadt, Germany) was added to the cell pellet, which was then immediately snap frozen in -80 celsius. Total RNA of TRIzol samples was extracted and purified using the RNeasy Mini Kit (217004, Qiagen, Valencia, CA), under fully automated sample preparation by the assistance of the QIAcube device (9001292, Qiagen, Valencia CA) following the manufacturer’s protocol. After extraction, total RNA yield and quality were evaluated using NanoDrop at 260 nm and the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA)
Label
Cy3
Label protocol
Labeling reactions were performed for each sample using Agilent Low Input Quick Amp Labeling, One-Color (Agilent Technologies, Santa Clara, CA). Briefly, with a starting concentration of 100 nanograms of total RNA, an initial cDNA strand was synthesized using a oligo(dT)24 primer containing a T7 RNA polymerase, this cDNA was then used as a template to generate Cy3 labeled cRNA by a reverse transcriptase enzyme. After the cRNA was obtained a purification step was performed using RNeasy Mini Kit (74104, Qiagen, Valencia, CA) followed by measurement of the yield and specific activity of each sample
Hybridization protocol
1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ml of 2x GEx Agilent hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays, 4x44K (Freiburg and Siena) or 8 x 60K (Leuven) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA High Resolution Microarray Scanner (G2505C US45102918) using one color scan setting for 8 x 60K array slides (Freiburg and Siena) and 8 x 60K array slides (Leuven). Scan Area 61x21.6 mm, Scan resolution 5 μm, Tiff 20 bit, Dye channel is set to Green.
Description
1011
Data processing
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Gene expression data was normalized to the geometric mean of all the IPF samples and controls using quantile normalization. Non-expressed and invariant transcripts were removed. For statiscal analysis we used the average gene expression values of the replicated probes. After merging the datasets of the 3 cohorts we obtained a final dataset of 20330 transcript measurements across the 176 IPF samples and 20 healthy donor samples.
