disease: PDAC patient with long (> 2 years) survival
Extracted molecule
total RNA
Extraction protocol
Total RNA, including miRNA, was extracted from archival sera (600 ul) of 15 PDAC patients and from 4 non cancer subjects (controls) by miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. miRNAs were quantified by Qubit microRNA assay kit (Thermo Fisher Scientific, Waltham, Massachusetts, United States). RNA integrity and the content of microRNAs (%) in each sample were assessed by capillary electrophoresis with the RNA 6000 Nano LabChip and the Small RNA Nano LabChip, respectively, using the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Only samples with RNA Integrity Number (R.I.N.) values higher than 6 and and quantity ≥4000 pg were used for microarray. analysis.
Label
Cy3
Label protocol
Total RNA (4.000 pg) was labeled with pCp Cy3, according to the Agilent’s protocol and unincorporated dyes were removed with MicroBioSpin6 columns (BioRad, Hercules, CA, USA).
Hybridization protocol
Probes were hybridized at 55°C for 22 hours using the Agilent's hybridization oven that is suited for bubble-mixing and microarray hybridization processes.
Scan protocol
Slides were scanned on an Agilent microarray scanner (model G2565CA) at 100% and 10% sensitivity settings. Agilent Feature Extraction software version 12.0.0.7 was used for image analysis.
Description
Circulating miRNA expression profiling of PDAC patient with long survival (pz 585)
Data processing
Inter-array normalization of miRNA expression levels was performed with cyclic Lowess for miR [Risso et al., 2009] and the average of replicates was used. Feature Extraction software (Agilent Technologies) was used to obtain spot quality measures in order to evaluate the quality and the reliability of the hybridization. In particular, the flag “glsFound” (set to 1 if the spot has an intensity value significantly different from the local background, 0 otherwise) was used to filter out unreliable probes: flag equal to 0 will be noted as “not available” (NA). So, in order to perform a more robust and unbiased statistical analysis, probes with a high proportion of NA values were removed from the dataset. NA (47%) was used as threshold in the filtering process, obtaining a total of 142 available human miRNAs.
