Lysis and homogeneization is performed with 1 mL pre-warmed (60°C) TRIzol Reagent (Invitrogen Cat. No. 15596-026) and 5 mm stainless steel beads (Qiagen Cat. No. 69989, 2 times 2 min at 25 Hz in tissue lyser Mixer Mill 300 for 2-3 mm Ø biopsies). The extraction is completed by adding Phase Lock Gel (Eppendorf Cat. No. 0032007.970) during the phase extraction. Molecular biology grade glycogen (Roche Diagnostics GmbH, Cat. N. 901393) is added to the aqueous phase during the precipitation step. After purification step on RNeasy Microkit columns (Qiagen, Cat. No. 74004), the final cleaned total RNA is eluted in 14 µL of RNase free water and stored at -80°C. RNA levels, quality and purity are assessed with the use of RNA 6000 Nano and Pico assay on the Agilent 2100 Bioanalyzer.
Label
Cy5
Label protocol
Commercial human reference RNA (Stratagene) was labeled per protocol and materials of the Ambion labelling kit (#1753). Labeling of RNA samples will be based on Ambion Amino Allyl MessageAmp II aRNA Amplification Kit (#q1753). 50 ng of total RNA was used for each labeling. The first step is a reverse transcription to synthesize first-strand cDNA with an oligo(dT) primer with a T7 promoter. Subsequently, the second-strand cDNA is synthesized. The cDNA was used for processing with the Ambion kit according to the protocol as template for in vitro transcription (IVT) with T7 RNA polymerase. During this amplification step, 5-(3- aminoallyl)-UTP (aaUTP) is incorporated into the cRNA. The resulting amplified RNA (aRNA) is then chemically coupled to N-hydroxysuccinimidyl ester-derivatized Cy5-dyes (NHS ester Cy5-dyes). Once purified, concentration and yield/dye incorporation are determined. The labeled products are aliquoted and stored at -80°C for further use
Hybridization protocol
A Tecan HS 4800 Hybridization station from Tecan, Inc. is used for prewash/hybridization/postwash of the microarrays. Prewash consists of 3 cycles with wash buffer at RT/75°C/50°C (each 20 s) followed by an additional 10 s wash with hybridization buffer at 50°C. One hundred L sample (100 ng labeled RNA) in hybridization buffer is then injected into the flow chambers at 50°C and agitated for 1 min. Subsequently, after 10 min at 75°C, the temperature is adjusted to 42°C for 16 h (agitation). The postwash consists in 4 cycles wash buffer at 42°C followed by an additional wash at 23°C (each 20 s). Finally, the slides are washed 3 times with diluted wash buffer at RT, dried in N2 stream, and scanned immediately with an Agilent LS fluorescence scanner (gain 80%).
Scan protocol
Agilent laser scanner at 100% gain, 10micron resolution, Cy5 channel. Files stored as 16bit TIF.
Description
Tissue samples from a series of 41 chemotherapy-naïve non-small cell lung cancer patients and 15 control patients with inflammatory lung diseases.
Data processing
The fluorescence images is analyzed by means of Array Pro (Mediacybernetics, Inc. US). Net signals is calculated by subtracting the 75 percentile of local corner background from trimmed mean of each spot (20% trimming on both sides of the intensity histogram). Individual samples are processed on separate slides in single color mode (Cy5). The 75 percentile of intensity histogram is adjusted to 200 counts (a.u.) for normalization of the microarrays. The image analysis is configured in a way that allowed an automated assessment of the quality of individual fluorescence spots (criteria: local fluorescence background, spots symmetry, presence in salt image/Print QC). Only microarrays with >98% qualified spots are used for further analysis.
