tissue: plasma disease: lung cancer patient id: P timepoint: 2
Treatment protocol
Blood of lung cancer patients was drawn directly before tumor resection (TP1), ~ two weeks after tumor resection (TP2) and then ~ three months (TP3), six months (TP4), nine months (TP5), 12 months (TP6), 15 months (TP7) and 18 months (TP8) after tumor resection. Stored at -20 degree
Extracted molecule
total RNA
Extraction protocol
We first treated 100µl plasma with 10µg Heparinase I (Sigma) and 100U RNaseOUTTM (Life Technologies, CatNo 10777-019) and incubated the mixture at 25°C for 1 hour. Nuclease free water (Life Technologies, CatNo AM9932) was added to a final volume of 250µl. A total of 750µl TRIzolLS (Life Technologies, CatNo 10296-028) was added and incubated at RT for 5 min. Then, 20µg glycogen, 5µl spike-in miRNA (miRNA mimic syn-cel-miR-39, 5nM, Qiagen), and 200µl chloroform were added and incubated for 3 min at RT. After centrifugation at 14000rpm and 4°C, the watery phase was transferred into a new tube and RNA was precipitated with 1,5 volumes of 100% ethanol. RNA was then isolated using the miRNeasy Mini Kit (Qiagen, CatNo 217004) according to manufacturers instructions but with the use of the RNeasy Mini Elute column to allow for a reduced elution volume of 15µl.
Label
Cy3
Label protocol
A total of 100 ng total RNA was processed using the miRNA Complete Labeling and Hyb Kit (Agilent, CatNo 5190-0456) to generate fluorescently (cyanine-3) labeled miRNA
Hybridization protocol
The microarrays, that contain 40 replicates of each of the 1,205 miRNAs of miRBase v16 (http://www.mirbase.org/) were hybridized with the labeled miRNA for 20 hours at 55°C and 20rpm.
Scan protocol
Agilent C Scanner, Agilent Scan Control Software
Data processing
Feature Extraction Software, summarizing of technical replicates by median and quantile normalization. Batch effect correction with ComBat R package.
